A review of non-prostanoid, eicosanoid receptors: expression, characterization, regulation, and mechanism of action

Abstract

Eicosanoid signaling controls a wide range of biological processes from blood pressure homeostasis to inflammation and resolution thereof to the perception of pain and to cell survival itself. Disruption of normal eicosanoid signaling is implicated in numerous disease states. Eicosanoid signaling is facilitated by G-protein-coupled, eicosanoid-specific receptors and the array of associated G-proteins. This review focuses on the expression, characterization, regulation, and mechanism of action of non-prostanoid, eicosanoid receptors.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12079-021-00630-6.

Introduction

Eicosanoids are signaling molecules derived from the oxidation of polyunsaturated fatty acids (PUFA). The term eicosanoid derives from the ancient Greek word eikosi, referring to the number 20 and was originally used to refer to lipids similar in structure to the twenty-carbon arachidonic acid. The term has been since expanded to include shorter and longer chain fatty acids that are also involved in signaling. They play a number of biological roles including initiation and resolution of inflammation, blood flow and blood pressure homeostasis, as well as pain perception. They also play a role in the progression of many disease states. These short-lived biomolecules typically act as autocrine or paracrine signaling agents for which their metabolic products may also serve as signaling molecules in their own right. This review is an extension of a companion article (Biringer 2020) with a focus on signaling by the non-prostanoid eicosanoids. The focus of this manuscript is primarily on human receptors for these eicosanoids but includes data for other organisms with significant sequence similarity when the data for humans is lacking.

Eicosanoid signaling occurs through specific G-protein coupled receptors (see Figs. 1 and 2). Eicosanoid receptors are typically multi-pass, heptahelical membrane proteins, belonging to the G-protein coupled receptor 1 family (GPCR) of proteins. These are by far amongst the most abundant membrane proteins known (Binda et al. 2014). The biological result of eicosanoid action on cells is not only cell type and tissue dependent, but also dependent on which eicosanoid is acting on a particular receptor. Eicosanoid action diversity is also defined by the diversity of G-proteins that most receptors are able to couple to, leading to actuation of different signaling pathways by the same receptor (Table 1). Some receptors have the capability to form heterodimers with other receptors and in doing so modify their own or the hetero-partner’s activity. In contrast to this diversity of action, many receptors show commonalities in their regulation. Most eicosanoid receptors show agonist-induced desensitization that is usually accompanied by receptor phosphorylation by various protein kinases. Often times phosphorylation leads to uptake into punctate vesicles through a variety of different mechanisms for temporary storage or degradation.

Fig. 1.

Metabolic pathways for non-prostanoid eicosanoids derived from arachidonic acid and their receptors. Gene designations are given for the participating enzymes (rounded boxes), accepted acronyms are given for metabolites (ovals) and accepted receptor acronyms in grey squares. Black arrows indicate enzymatic reactions and grey arrows indicate receptor binding. Enzymes are as follows: 12/15-LOX, 12/15-lipoxygenase; 5-HEDH, 5-hydroxyeicosanoid dehydrogenase; ALOX12, Arachidonate 12(S)-lipoxygenase; ALOX12B, arachidonate 12(R)-lipoxygenase; LTC4S, leukotriene C4 synthase; ALOX15, arachidonate 15-lipoxygenase-1; ALOX15B, arachidonate 15-lipoxygenase-2; ALOX5, Arachidonate 5-lipoxygenase; ALOXE3, arachidonate lipoxygenase 3; CYP2C, cytochrome P450 C2; CYP4F2, cytochrome P450 4F; CYP2J, cytochrome P450 J2; DPEP, dipeptidase; GPX, glutathione peroxidase; LTA4H, Leukotriene A-4 hydrolase; LTC4S, leukotriene C4 synthase; PTGS1, Prostaglandin G/H Synthase 1; PTGS2, Prostaglandin G/H Synthase 2; PTGS2/aspirin, PTGS2 acetylated by aspirin; sEH, soluble epoxide hydrolase; TBXAS1, Thromboxane A Synthase 1. Abbreviations for metabolites: 8,9-EET, 8,9-EET, 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid; 11,12-EET, 11,12-epoxy-5Z,8Z,14Z-eicosatrienoic acid; 14,15-EET, 14,15-epoxy-5Z,8Z,11Z-eicosatrienoic acid; 11(R),12(R)-HXA₃, 11(R),12(R)-Hepoxilin A₃; 11(S),12(S)-HXA₃, 11(S),12(S)-Hepoxilin A₃; 11(S),12(S)-HXB₃, 11(S),12(S)-Hepoxilin B₃; 11(R),12(R)-TrXA₃, 11(R),12(R)-trioxilin A₃;11(S),12(S)-TrXA₃, 11(S),12(S)-trioxilin A₃; 11(S),11(S)-TrXB₃, 11(S),12(S)-trioxilin B₃; 5-oxo-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; 12(S)-HETE, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 12(S)-HPETE, 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 5(S)-HETE, 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5(S)-HPETE, 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 15(S)-HPETE, 15S-hydroperoxy-5Z,8Z,11Z,13E- eicosatetraenoic acid; 15(S)-HPETE, 15S-hydroperoxy-5Z,8Z,11Z,13E- eicosatetraenoic acid; 15(R)-HETE, 15R-hydroxy-5Z,8Z,11Z,13E- eicosatetraenoic acid;12(R)-HPETE, 12R-hydroperoxy-5Z,8Z,10E,12R,14Z- eicosatetraenoic acid; 12(R)-HETE, 12R-hydroxy-5Z,8Z,10E,12R,14Z- eicosatetraenoic acid; 12-KETE, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid;12-HHT, 12-Hydroxy-5,8,10-heptadecatrienoic acid; AA, arachidonic acid; 20-HETE, 20-Hydroxyeicosatetraenoic acid; 5(S),6(S)-Ep-15(R)-HETE, 5S,6S-epoxy-15(R)-hydroxy-7E,9E,11Z,13E-eicosatetraenoic acid; 5(S),6(S)-Ep-15(S)-HETE, 5S,6S-epoxy-15(S)-hydroxy-7E,9E,11Z,13E-eicosatetraenoic acid; 15-epi-LXA₄, 15-epi-lipoxin A₄; 15-epi-LXB₄, 15-epi-lipoxin B₄; LTA₄, leukotriene A₄; LTB₄, leukotriene B₄; LTC₄, leukotriene C₄; LTD₄, leukotriene D₄; LTE₄, leukotriene E₄; LXA₄, lipoxin A₄; LXB₄, lipoxin B₄; EXA₄, eoxin A₄; EXC₄, eoxin C₄; EXD₄, eoxin D₄; EXE₄, eoxin E₄; PGH₂, prostaglandin H₂. Receptors are as follows: CYSLTR1, cysteinyl leukotriene receptor 1; CYSLTR2, cysteinyl leukotriene receptor 2; FFAR1, free fatty acid receptor 1; FPR2/ALX, N-formyl peptide receptor 2; GPR17, uracil nucleotide/cysteinyl leukotriene receptor; GPR31, 12S-hydroxy-5,8,10,14-eicosatetraenoic acid receptor; GPR32, Probable G-protein coupled receptor 32; GPR75, Probable G-protein coupled receptor 75; LTB4R, Leukotriene B₄ receptor 1; LTB4R2, leukotriene B₄ receptor 2; OXER1, oxoeicosanoid receptor 1; OXGR1 (GPR99), 2-oxoglutarate receptor 1; P2Y12, P2Y purinoceptor 12; TP, Thromboxane A₂ receptor (TBXAR2); TRPV1, Transient receptor potential cation channel subfamily V member 1

Fig. 2.

Metabolic pathways for non-prostanoid eicosanoids derived from eicosapentaenoic acid (EPA) and dicosahexaenoic acid (DHA). Gene designations are given for the participating enzymes (rounded boxes), accepted acronyms are given for metabolites (ovals) and accepted receptor acronyms in grey squares. Black arrows indicate enzymatic reactions and grey arrows indicate receptor binding. Enzymes are as follows: ALOX, unspecified lipoxygenase; ALOX15, arachidonate 15-lipoxygenase-1; ALOX5, Arachidonate 5-lipoxygenase; GPX, glutathione peroxidase; Hyd, unspecified hydrolase; P450, unspecified P450 enzyme; PTGS2/aspirin, PTGS2 acetylated by aspirin. Abbreviations for metabolites: 11(R),12(R)-HXA₃, 11(R),12(R)-Hepoxilin A₃; 11(R),12(R)-TrXA₃, 11(R),12(R)-trioxilin A₃; 11(R),12(S)-TrXB₃, 11R,12(S)-trioxilin B₃; 11(S),12(S)-HXA₃, 11(S),12(S)-Hepoxilin A₃; 11(S),12(S)-HXB₃, 11(S),12(S)-Hepoxilin B₃; 11(S),12(S)-TrXA₃, 11(S),12(S)-trioxilin A₃; 12(S)-HETE, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 12(S)-HPETE, 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 15(R)-HETE, 15R-hydroxy-5Z,8Z,11Z,13E- eicosatetraenoic acid; 15(S)-HPETE, 15S-hydroperoxy-5Z,8Z,11Z,13E- eicosatetraenoic acid; 15(S)-HPETE, 15S-hydro peroxy-5Z,8Z,11Z,13E- eicosatetraenoic acid; 17(R)-HDHA, 17R-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid; 17(R)-HPDHA, 17R-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid; 17(R)-RvD1, 17(R)-resolvin D1; 17(R)-RvE2, 17(R)-resolvin E2; 17(S)-HDHA, 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid; 17(S)-HPDHA, 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid; 17(S)-HPDHA, 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid; 17(S)-RvD1, 17(S)-resolvin D1; 17(S)-RvD2, 17(S)-resolvin D2; 17(S)-RvE2, 17(S)-resolvin E2; 18(R)-HEPE, 18R-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid; 18(R)-HPEPE, 18R-hydroperoxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid; 18(R)-RvE2, 18(R)-resolvin E2; 18(R)-RvE2, 18(R)-resolvin E2; 18(S)-HEPE, 18S-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid; 18(S)-HPEPE, 18S-hydroperoxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid; 18(S)-RvE1, 18(S)-resolvin E1;18(S)-RvE2, 18(S)-resolvin E2; 5(S),6(S)-Ep-18(R)-HEPE, 5S,6S-epoxy,18R-hydroxy-7E,9E,11Z,14Z,16E-eicosapentaenoic acid; 5(S),6(S)-Ep-18(S)-HEPE, 5S,6S-epoxy,18S-hydroxy-7E,9E,11Z,14Z,16E-eicosapentaenoic acid; 5(S)-Hp-18(R)-HEPE, 5S-hydroperoxy-18R-hydroxy-(6E,8Z,11Z,14Z,16E)-icosapentaenoate; 5(S)-Hp-18(S)-HEPE, 5S-hydroperoxy-18S-hydroxy-(6E,8Z,11Z,14Z,16E)-icosapentaenoate; 5-oxo-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5S-HETE, 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5(S)-HPETE, 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 7(S),8(S)-Ep-17(R)-HDHA, 7S,8S-epoxy-17R-hydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic; 7(S),8(S)-Ep-17(S)-HDHA, 7S,8S-epoxy-17S-hydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic; DHA, docosahexaenoic acid; EPA, Eicosapentaenoic acid. Receptors are as follows: CMKLR1, Chemokine-Like Receptor 1; FPR2/ALX, N-formyl peptide receptor 2; GPR18, N-Arachidondyl Glycine Receptor; GPR32, G-Protein Receptor 32

Table 1.

Signaling pathways and G-protein association for Eicosanoid receptors

Receptor	signaling pathway	G-protein mediation	Reference
GPR31	IP3↑ Ca2+↑	Gαq	Guo et al. (2011)
	cAMP↓	Gαi	Mashiko et al. (2019)
	rho	Gα11/13	Mashiko et al. (2019)
LTB4R2	IP3↑ Ca2+↑	Gαi	Okuno et al. (2008); Arcemisbéhère et al. (2010)
	IP3↑ Ca2+↑	Gαq	Okuno et al. (2008); Arcemisbéhère et al. (2010)
OXER1	IP3↑ Ca2+↑	Gβγ	Hosoi et al. (2002, 2005); Jones et al. (2003
	IP3↑ Ca2+↑	Gαi	Hosoi et al. (2002, 2005); Jones et al. (2003)
GPR75	IP3↑ Ca2+↑	Gαq11	Garcia et al. (2017)
FFAR1/GPR40	IP3↑ Ca2+↑	Gαq/Gβγ	Itoh et al. (2003); Briscoe et al. (2003)
	IP3↑ Ca2+↑	Gαi	Itoh et al. (2003); Briscoe et al. (2003)
TRPV1	Ca2+↑, Na+↑	none	Marsh et al. (1987)
LTB4R	cAMP↓	Gαi	Saeki and Yokomizo (2017); Malfacini et al. (2019); Kuniyeda et al. (2007)
	IP3↑ Ca2+↑	Gαq	Saeki and Yokomizo (2017); Malfacini et al. (2019); Kuniyeda et al. (2007)
	IP3↑ Ca2+↑	Gα16	Saeki and Yokomizo (2017); Malfacini et al. (2019); Kuniyeda et al. (2007)
CYSLTR1	Ca2+↑	Gαq11,	Capra et al. (2003)
	Ca2+↑	Gαio	Capra et al. (2003)
	Ca2+↑	Gβγ	Capra et al. (2003)
	cAMP↓ Ca2+↑	Gαq	Parmentier et al. (2012)
	cAMP↓ Ca2+↑	Gαi	Parmentier et al. (2012)
	Ca2+↑	Gαi3	Adolfsson et al. (1996)
CYSLT2R	IP3↑ Ca2+↑	Gαq11	Sarau et al. (1999); Moore et al. (2016)
	cAMP↓	Gαio	Mellor et al. (2003)
OXGR1	cAMP↑	Gαq	Inbe et al. (2004)
	cAMP↑	Gαs	Inbe et al. (2004)
	Ca2+↑	Gαq	Steinke et al. (2014); He et al. (2004)
GPR17	cAMP↓	Gαi	Hennen et al. (2013); Simon et al. (2015)
	cAMP↑	Gαs	Hennen et al. (2013); Simon et al. (2015)
	IP3↑ Ca2+↑	Gαq	Hennen et al. (2013); Simon et al. (2015)
P2Y12	Ca2+↑	Gαi	Soulet et al. (2004)
	IP3↑	Gαi	Bodor et al. (2003)
	cAMP↓	Gαi	Kauffenstein et al. (2004)
FPR2/ALX	Ca2+↑	Gαi	Le et al. (2002)
	cAMP↓ Ca2+↑	Gαi	Ge et al. 2020
GPR18	Ca2+↑	Gαi	Kohno et al. 2006
GPR32	Ca2+↑	Gαi/o	Hodges et al. (2013)
CMKLR1a	Ca2+↑	Gαi	Arita et al. (2005), Wittamer et al. (2004)

^aExperimental evidence for chemerin signaling is available, but only indirect support for RvE1 signaling is found (see text)

ETE, HETE and oxidized HETE receptors

Introduction

The unstable hydroperoxyeicosatetraenoic acids (HPETE) and their hydroxyeicosatetraenoic acid (HETE) reduction products are notable not only for their function in signaling, but also as precursors for lipoxins, eoxins, leukotrienes, and various other oxidized PUFA products (Brash, 1999). As an example, 15(S)-HPETE and 12(S)-HETE are involved in cell survival mechanisms (Tang et al. 1996) and the binding of monocytes to vascular tissue (Natarajan and Nadler, 2004; Sultana et al. 1996). Both 5-HPETE and 12(S)-HPETE are also involved in modulating neurotransmission (Piomelli et al. 1987). Further, the oxidized derivative of the former, 5-oxo-hydroxyeicosatetraenoic acid (5-oxo-ETE), is a notable activator of neutrophils (Powell et al. 1993). These oxidized derivatives of arachidonic acid are formed enzymatically or through non-enzymatic lipid peroxidation mechanisms (review, Powell and Rokach 2015).

GPR31

Introduction

Human 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) receptor (hGPR31) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds various hydroxyeicosatetraenoic acids with a particular affinity for 12(S)-HETE which stimulates the mitogen-activated protein kinase (MAPK) pathway leading to activation of signal-regulated kinase 1 and 2 (ERG1/2), MAPK/ERK kinase (MEK), and the NF-κB transcription factor (Guo et al. 2011). GPR31 is intimately involved in pro-inflammatory processes as well as cell mobility and long-term cell survival (Guo et al. 2011; Fretland et al. 1995). This receptor is also activated through proton binding, thus serving as an extracellular proton sensor (Goldsmith and Dhanasekaran, 2007; Mashiko et al. 2019).

hGPR31 (UniProtKB-O00270) is translated as a 319 amino acid polypeptide with a calculated molecular weight of 35.1 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 16 residues and a cytosolic C-terminal with 34 residues. There are no additional isoforms and one known coding single nucleotide polymorphic (SNP) variant (H91R) for which no functional abnormalities have been reported (Zingoni et al. 1997; https://genecards.org, Stelzer et al. 2016; Landrum et al. 2016https://www.ncbi.nlm.nih.gov/clinvar/). There are no reported X-ray structures, however, there is one model available on the SWISS-Model site using P2Y purinoceptor (27.8% sequence homology; template PDB entry 4XNV) to serve as a working model (https://swissmodel.expasy.org/, Waterhouse et al. 2018).

There are several N- and O-glycosylations predicted by sequence, but none are confirmed experimentally. Here, and for all receptors discussed in this document, N-glycosylation was predicted using NetNGlyc analysis (http://www.cbs.dtu.dk/services/NetNGlyc/, Blom et al. 2004, Steentoft et al. 2013) and O-glycosylation was predicted using NetOGlyc analysis (http://www.cbs.dtu.dk/services/NetOGlyc/, Steentoft et al. 2013). There are numerous potential phosphorylation sites on the hGPR31 receptor based on motifs, but none are specifically confirmed experimentally. Phosphorylation was predicted for this and the other receptors discussed here using the NetPhos 3.1 server (http://www.cbs.dtu.dk/services/NetPhos/, Blom et al. 1999), a conservative minimum score of 0.9, and the availability of sites based on topology predictions (UniProtKB) for all but GRK kinases. GRK phosphorylation sites are predicted using the GPS server (http://gps.biocuckoo.cn/, Xue et al. 2011). Predicted posttranslational modifications for this receptor and all others discussed here are given in the supplement, Table S1.

Expression and characterization

The hGPR31 receptor is expressed primarily in the immune system, but is also found in the gastrointestinal tract, and male- and female-specific tissues (http://proteinatlas.org; Uhlén et al. 2015; Town et al. 1983; Sharif et al. 2000).

Ligand binding properties for GPR31 have been characterized with human recombinant protein and recombinant GPR31-bovine Gα_i fusion protein expressed in Chinese hamster ovary (CHO) cells (Fig. 1, Table 2). 12(S)-HETE binds tightly (K_d = 4.8 nM) in a regio- and stereo-specific manner with much higher signaling efficiency than either 15(S)-HETE and 5(S)-HETE (Guo et al. 2011). In addition to 12(S)-HETE activation, protons also stimulate GPR31. Proton-induced signaling exhibits an EC₅₀ equivalent to pH 5.6 for a GPR31-Gα_i fusion protein and around pH 5.8 for the recombinant GPR31 receptor alone (Mashiko et al. 2019).

Table 2.

ETE, HETE and oxidized HETE receptors

Receptor/Expression	Parameter measured	12(S)-HETE	12(S)-HPETE	12(R)-HETE	15(S)-HETE	5(S)-HETE	5(S)-HPETE	5-OXO-ETE	5,15-di-HETE	LTB4	12-HHT	RvE1	Reference
Ki (nM)
hGPR31/CHOa	Disp	4	–	–	–	–	–	–	–	–	–	–	Guo et al. (2011)
hLTB4R2/CHOb	Disp	4000	4000	6000	9000	–	–	–	–	180	–	–	Yokomizo et al. (2001)
hLTB4R2/CHOc	Disp	–	–	–	–	–	–	–	–	21	2.3	–	Okuno et al. (2008)
hLTB4R2/monomerd	Disp	–	–	–	–	–	–	–	–	25	3.7	–	Arcemisbéhère et al. (2010)
hLTB4R2/dimerd	Disp	–	–	–	–	–	–	–	–	21	4.4	–	Arcemisbéhère et al. (2010)
hLTB4R/COS-7	Disp	6000a	–	30	–	–	–	–	–	0.38	–	–	Yokomizo et al. (1997)
hLTB4R/HEKe	Disp	–	–	–	–	–	–	–	–	0.08	–	34.3	Serhan et al. (2011)
hLTB4R/HEKe	Disp	–	–	–	–	–	–	–	–	3	–	70	Arita et al. (2007)
hOXER1/PMNf	Disp	–	–	–	970	–	–	4	61	–	–	–	O'Flaherty et al. (1998)
EC50/IC50 (nM)
hGPR31/CHOg	GTPγS	0.28	–	–	42	386	–	–	–	–	–	–	Guo et al. (2011)
hGPR31-Sf9/memh	GTPγS	100	–	–	–	–	–	–	–	–	–	–	Mashiko et al. (2019)
hLTB4R2/CHOi	Ca2+↑	5000	–	–	> 10,000	–	–	–	–	700	–	–	Yokomizo et al. (2001)
hLTB4R2/CHOj	Chemo	5000	–	–	20,000	–	–	–	–	60	–	–	Yokomizo et al. (2001)
hLTB4R2/CHOi	Ca2+↑	–	–	–	–	–	–	–	–	142	19	–	Okuno et al. (2008)
hLTB4R2/CHOi	Ca2+↑	–	–	–	–	–	–	–	–	200	5	–	Okuno et al. (2015)
hLTB4R2/mBMMCk	GTPγS	5000	–	–	–	–	–	–	–	–	–	–	Lundeen et al. (2006)
mLTB4R2/300.19 l	Ca2+↑	–	–	–	–	–	–	–	–	20	6.3	–	Mathis et al. (2010)
hLTB4R2/monomerm	GTPγS	–	–	–	–	–	–	–	–	70	–	–	Arcemisbéhère et al. (2010)
hLTB4R2/dimerm	GTPγS	–	–	–	–	–	–	–	–	70	–	–	Arcemisbéhère et al. (2010)
hLTB4R/COS-7n	Ca2+↑+	–	–	–	–	–	–	–	–	10	–	–	Yokomizo et al. (1997)
hLTB4R/COS-7 m	GTPγS	–	–	–	–	–	–	–	–	5	–	–	Kuniyeda et al. (2007)
hLTB4R/CHOn	Ca2+↑	–	–	–	–	–	–	–	–	3	–	–	Okuno et al. (2015)
hLTB4R/CHOo	cAMP↓	–	–	–	–	–	–	–	–	11.5	–	3.2	Serhan et al. (2011)
hLTB4R/HEKo	cAMP↓	–	–	–	–	–	–	–	–	0.015	–	3.2	Arita et al. (2007)
hOXER1-Gαi1/CHOp	GTPγS	–	–	–	–	240	69	5.7	–	–	–	–	Hosoi et al. (2002)
hOXER1/CHOq	cAMP↓	–	–	–	–	–	–	33	–	–	–	–	Hosoi et al. (2002)
hOXER1/CHOi	Ca2+↑	–	–	–	–	510	98	5.1	–	–	–	–	Hosoi et al. (2005)
hOXER1/HEKq	cAMP↓	–	–	–	–	–	–	0.33	–	–	–	–	Jones et al. (2003)
hOXER1/PMNr	Ca2+↑	–	–	–	6300	–	–	13	100	–	–	–	O'Flaherty et al. (1998)
hOXER1/Neui	Ca2+↑	–	–	–	–	150	–	2	900	0.2	–	–	Powell et al. (1993)
hOXER1/Eoss	Migration	–	–	–	–	–	–	20	–	–	–	–	Powell et al. (1995)

Agonist parameters given for Human (h) and murine (m) receptors in the indicated cell line or oligomeric state. Parameter measured: Ca²⁺↑, increase in [Ca²⁺]_i; cAMP↓, decrease in forskolin induced cAMP accumulation; chemo, chemotactic response; disp, displacement assay; GTPγS, binding of [³⁵S] GTPγS; migration, eosinophil migration; Cell type abbreviations: 300.19, Abelson-transformed murine pre-B lymphoma; BMMC, bone marrow mononuclear cells; CHO, Chinese hamster ovary cells; COS-7, African green monkey kidney cell line; Eos, eosinophils; HEK, Human embryonic kidney 293 cells; Neu, neutrophils; PMN, human polymorphonuclear leukocytes; Sf9, clonal isolate of Spodoptera frugiperda Sf21 cells. Agonist abbreviations: 12(S)-HETE, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 12(S)-HPETE, 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 12(R)-HETE, 12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 15(S)-HETE, 15(S)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; 5(S)-HETE, 5S-Hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5(S)-HPETE, 5S-Hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5-OXO-ETE, 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5,15-di-HETE, 5,15-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid; LTB₄, leukotriene B₄; 12-HHT, 12-hydroxy-5E,8E,10E-heptadecatrienoic acid; RvE1, resolvin E1. a) Estimated from graph for displacement of 12(S)-[³H]HETE and K_i calculated using the Cheng-Prusoff equation. b) Calculated from the IC₅₀ estimated from a graph for displacement of [³H]LTB4 using Cheng-Prusoff equation for human BLT2 expressed in CHO cells. c) Calculated for displacement of [³H]LTB₄ using Cheng-Prusoff equation for human BLT2 and a K_d of 23 (Yokomizo et al. 2001) expressed in CHO. d) Calculated from IC₅₀ for displacement of fluorescent labeled LTB₄ using Cheng-Prusoff equation. e) Calculated for displacement of [³H]RvE1. f) Calculated from IC₅₀ estimated from graph for displacement of [³H]-5-oxo-ETE using Cheng-Prusoff equation; OXER1 is the most likely receptor in polymorphonuclear neutrophils (PMN). g) Measured with [³⁵S]-GTPγS binding assay. h) Determined with a [³⁵S]GTPγS binding assay of membranes from Sf9 cells expressing human GP31-bovine G1a fusion protein. i) Measured intracellular Ca²⁺ increase. j) Chemotactic response. k) Chemotaxis for mouse bone marrow derived mast cells. Note: BLT1 and BLT2 receptors are present, but only BLT2 receptors bind 12-HETE. l) Ca²⁺ release in murine 300.19 cells. m) Measured with [³⁵S]-GTPγS binding assay in the presence of Gα_i. n) Estimated from graph for increasing cytosolic Ca²⁺. o) Calculated from the inhibition of forskolin-activated cAMP. p) Determined with a [³⁵S]-GTPγS binding assay from a fusion protein expressed in CHO cells and EC₅₀. 5(S)-HPETE binding estimated from the data curve. q) Determined from a reduction in forskolin-induced cAMP production. r) Estimated from graph for increasing cytosolic Ca²⁺; OXER1 is the most likely receptor in polymorphonuclear neutrophils (PMNs) determined from eosinophil migration

Mechanism of cell activation

The binding of 12(S)-HETE to GPR31 activates protein kinase C-a (PKC-a) and does so through an inositol 1,4,5-trisphosphate (IP₃) mediated pathway (Guo et al. 2011) which is consistent with signaling through the G-protein Gα_q. This result and additional studies show that 12(S)-HETE ultimately leads to activation of NF-κB, MEK and ERK1/2 by PKC-dependent and -independent mechanisms, the latter involving the activation of Src kinases that lead to phosphorylation of adapter proteins (i.e. shc and Grb2) which in turn promotes activation ERK1/2 via the GTPase Ras (Guo et al. 2011; Liu et al. 1995; Szekeres et al. 2000). The involvement of Src and the additional observation that pertussin toxin inhibits activation of ERK1/2 suggests that signaling occurs through Gα_i as well. On the other hand, the naturally occurring the linoleic oxidation product OXLAM, 13-hydroxyoctadecadienoic acid (13-HODE) is a GPR31 antagonist with an IC₅₀ = 4 nM (Liu et al. 1995; Honn 2008). Protons can also activate this receptor in the absence of 12(S)-HETE with maximal activation below pH 5 via both Gα_i and Gα_11/13, suggesting a concomitant reduction of cAMP or a Rho-mediated signaling respectively (Mashiko et al. 2019). Interestingly, 12(S)-HETE stimulation of GPR31 in lymph endothelial cells leads to a 12-HETER/Rho/ROCK/MYPT signaling cascade that induces myosin light chain 2 (MLC2) function (Nguyen et al. 2016).

Regulation

There are no reports of regulation of GPR31 through phosphorylation or β-arrestin binding. This is likely due to the fact this orphan receptor was only recently shown to be the 12(S)-HETE receptor 12-HETER1 (Guo et al. 2011). A more recent report shows a profound up-regulation of 12-HETER1 in prostate cancer tumors and that it plays a critical role in cancer progression (Honn et al. 2016). Related results are observed in a murine hepatocellular carcinoma model where an increase in 12(S)-HETE serves to increase expression of GPR31 and that GPR31 promotes reoccurrence of hepatocellular carcinoma in nonalcoholic fatty liver disease (Yang et al. 2019).

LTB4R2

Introduction

Human leukotriene B4 receptor 2 (hLTB4R2) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is activated by various HETEs at micromolar levels, 12-Hydroxyheptadecatrienoic acid (12-HHT) at nanomolar levels, as well as various leukotrienes at somewhat higher levels (discussed in a later section). Stimulation of this receptor leads to signaling through MAPK and an increase in intracellular Ca²⁺ ([Ca²⁺]_i). LTB4R2 has a protective role in allergic airway inflammation and is involved in skin barrier function and wound healing.

hLTB4R2 (BL2, JULF2, UniProtKB-Q9NPC1) is translated as a 358 amino acid polypeptide with a calculated molecular weight of 37.9 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 24 residues and a cytosolic C-terminal with 62 residues. There are no additional isoforms and one reported coding SNP variant (P130S) with no reported effect on biological function (Landrum et al. 2016). There are no reported X-ray structures or models available. There is one potential structural model template, Leukotriene B4 receptor BLT1 in complex with BIIL260 (42.3% sequence homology; PDB entry 5X33, template 5 × 33.1.A), selected by the SWISS-MODEL website (Waterhouse et al. 2018) that can be used as a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

LTB4R2 is primarily expressed in the skin but is also found in other tissues in lower amounts, particularly in the immune and digestive systems (Uhlén et al. 2015). Although originally characterized as the low affinity LTB₄ receptor (Yokomizo et al. 2000, 2001) it has since been shown to be the primary receptor for 12-HHT (Fig. 1) which shows the tightest binding and lowest EC₅₀ of all agonists examined to date (Okuno et al. 2008, 2015) (Table 2). Interestingly, 12-HHT does not bind to the high affinity LTB₄ receptor (LTB4R), further supporting the notion that 12-HHT is the primary agonist for the LTB4R2 receptor (Okuno and Yokomizo 2018).

Mechanism of cell activation

12-HHT activation of LTB4R2 proceeds through both Gα_i and Gα_q mediated pathways, the former leading to a pertussis toxin-sensitive cAMP reduction and the latter to an increase in IP₃ and [Ca²⁺]_i (Okuno et al. 2008; Arcemisbéhère et al. 2010). It is also reported that 12(S)-HETE stimulation of LTB4R2 induces dose-dependent ERK signaling and changes in Protein kinase B (Akt, PKB) phosphorylation in murine mast cells (Lundeen et al. 2006). Park et al. (2019) reveal that 12-HHT stimulation of LTB4R2 in cultures of a KRAS mutant colorectal cancer cell line initiates the same pathways which results in the upregulation of cyclin D1 that in turn enhances the proliferation of these cells. This suggests that LTB4R2 may be a potential therapeutic target for KRAS mutant colorectal cancer. 12-HHT stimulation of overexpressed LTB4R2 transfected into human bronchial epithelial cells enhances migration and proliferation of these cells as well as enhancing the airway epithelial barrier integrity, suggesting that LTB4R2 may be a potential target for asthma treatment (Liu et al. 2018).

In the skin LTB4R2 is expressed on the surface of keratinocytes and is activated by 12-HHT produced by activated platelets. Activation by 12-HHT accelerates keratinocyte migration by inducing the production of tumor necrosis factor α (TNFα), interleukin (IL)-1β, and matrix metalloproteinases (MMPs), thus accelerating wound healing (Liu et al. 2014). Further, the 12-HHT/LTB4R2 axis enhances cell–cell junctions in both intestinal and skin epithelial cells by upregulating CLDN4, an integral membrane channel of tight junctions, and does so via a p38/MAPK signaling pathway (Saeki and Yokomizo 2017; Ishii et al. 2016). In addition, both 12(S)-HETE and 12-HHT recruit mast cells (Lundeen et al. 2006), another required function for wound healing (da Silva et al. 2014) and blood–brain barrier function (Kempuraj et al. 2019).

Regulation

Pretreatment of murine bone marrow mast cells with stem cell factor (SCF) downregulates expression of LTB4R2 thus reducing the cells’ chemotactic response to LTB4R2 agonists (Lundeen et al. 2006). LTB4R2 is upregulated in many cancers, particularly in highly aggressive forms, by mechanisms that are currently not understood (Kim et al. 2012; Seo et al. 2011, 2012). Regulation through phosphorylation, typical for G-protein receptors, has yet to be reported.

OXER1

Introduction

The human oxoeicosanoid receptor 1 (hOXER1) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds peroxy-, dihydroxy-, and oxo-eicosanoid derivatives of arachidonic acid. Stimulation of this receptor leads to activation of various phospholipase Cs (PLC), phosphoinositide 3-kinase (PI3K), ERK 1/2, and p38/MAPK and a reduction in cAMP.

hOXER1 (GPR170, GPR R527, GPR TG1019, GPCR48, UniProtKB-Q8TDS5) is translated as a 423 amino acid polypeptide with a calculated molecular weight of 46.0 kDa. The basic structure consists of seven transmembrane helices with a large N-terminal extracellular domain of 97 residues and a cytosolic C-terminal with 66 residues. There are no additional isoforms and four coding SNP variants reported: M316L (Stelzer et al. 2016), L407V (Jones et al. 2003), A368V, and T334P (Landrum et al. 2016). The biological ramifications of these SNPs are not reported. There are no reported X-ray structures. There is one model available on the SWISS-Model site using P2Y purinoceptor (23.4% sequence homology; PDB entry 4XNV) as a template (Waterhouse et al. 2018) that provides a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hOXER1 is expressed in most tissues with particularly prominent expression in the immune and digestive systems, as well as in liver, and male and female-specific tissues (Uhlén et al. 2015). OXER1 is the primary receptor for 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), produced enzymatically in several steps from AA (Fig. 1), with K_i and EC₅₀ values in the low nM range (Hosoi et al. 2002; Jones et al. 2003; O'Flaherty et al. 1998) (Fig. 1, Table 2). It also serves as a receptor for other oxidized AA derivatives, 5(S)-HPETE, 5,15-di-HETE, and 5(S)-HETE, with 10–100 times poorer efficiency than 5-oxo-HETE (Hosoi et al. 2002; Jones et al. 2003; O'Flaherty et al. 1998). 5-oxo-ETE serves as a chemoattractant for basophils, monocytes, neutrophils (Powell et al. 1993) and eosinophils, the effect on the latter being significantly greater than the others (Powell et al. 1995; Powell and Rokach 2013).

Mechanism of cell activation

5-oxo-ETE activation of OXER1 proceeds through both Gα_i- and Gβγ-mediated pathways (Hosoi et al. 2002, 2005; Jones et al. 2003) that lead to PLC/Ca²⁺ mobilization, and MEK/ERK and PI3K/Akt phosphorylation/activation when expressed in CHO cells (Hosoi et al. 2005). Stimulation of OXER1 in prostate cancer cells also promotes cell survival through PKCε that is activated by the diacylglycerol produced by OXER-1-activated PLC-β (Zingoni et al. 1997). Although not specifically stated, activation of PKCε by Ca²⁺ stimulated by the simultaneous production of IP₃ by PLC-β may also be involved.

5-oxo-ETE elicits various responses in human eosinophils, including a very rapid increase in [Ca²⁺]_i and actin polymerization (Czech et al. 1997; Powell et al. 1999), the latter is likely involved in cell migration (Mogilner and Oster 1996). Further, 5-oxo-ETE stimulates the release of the β-integrin CD11b and L-selectin in eosinophils, known mediators for lymphocyte adhesion and infiltration (Mogilner and Oster 1996). Lastly, 5-oxo-ETE also induces expression of urokinase plasminogen activator (uPAR) and secretion of metalloproteinase-9 (MMP-9), the former involved in tissue reorganization and the latter involved in the breakdown of extracellular matrix (Langlois et al. 2006). Taken together, these functions collectively promote the infiltration of eosinophils to the source of 5-oxo-ETE release (see also Powell and Rokach 2020).

Regulation

Typical G-protein regulation through phosphorylation or arrestin binding has not been reported to date. Regulation of OXER1 gene expression also has not been reported. However, androgens, testosterone in particular, antagonize the actions of 5-oxo-ETE on the OXER1 receptor expressed in DU-145 prostate cancer cells (Kalyvianaki et al. 2017, 2019). On the other hand, pretreatment of polymorphonuclear neutrophils (PMN) or eosinophils with granulocyte–macrophage-stimulating factor (GM-CSF) or granulocyte-stimulating factor (G-GSF) increases the potency of 5-oxo-ETE activation of OXER1 (O'Flaherty et al. 1996a, 1996b). The increased potency is manifested through an increase the 5-oxo-ETE-induced phosphorylation and hence activity of MAPKs (O'Flaherty et al. 1996b). The mechanism for this process is unknown.

GPR75

Introduction

The human GPR75 receptor (hGPR75) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is activated by the chemokine CCL5/RANTES and is a highly specific target for 20-HETE. The latter is one of the principal eicosanoids formed from AA by the action of cytochrome P450 enzymes (Fig. 1), in particular, the CYP4A and CYP4F families (Fan and Roman 2017). 20-HETE/hGPR75 axis plays a major role in renal, pulmonary, and cardiac function as well as vascular tone and inflammation (Fan et al. 2016; Roman et al. 2002) and is well established as a potent vasoconstrictor (Yu et al. 2003), particularly in cerebral and renal microvessels (Harder et al. 1995).

hGPR75 (GPR170, GPR R527, GPR TG1019, UniProtKB-O95800) is translated as a 540 amino acid polypeptide with a calculated molecular weight of 59.4 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 46 residues and a large cytosolic C-terminal with 169 residues. There are no additional isoforms and seven coding SNP variants (N78K, P99L, S108T, A116T, T135P, C160G, and L433V) (Sauer et al. 2001; Stelzer et al. 2016) with no reported effect on biological function. There are no reported X-ray structures. Only low quality models are calculated by the SWISS-MODEL website (Waterhouse et al. 2018) thus no working model is available. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hGPR75 is expressed in most tissues with particularly prominent expression in the brain, more in neuron-like cells than in astrocytes (Dedoni et al. 2018), and the endocrine system (Uhlén et al. 2015). In 2006 this receptor was identified as a RANTES/CCL5 chemokine receptor (Ignatov et al. 2006) and only recently as a receptor for 20-HETE. Both agonists show very tight binding with a measured K_i for 20-HETE of 0.1 nM (Garcia et al. 2017) and EC₅₀ values of 0.12 or 0.3 nM, depending on the cell type in which it is expressed (Fig. 1, Table 3) (Ignatov et al. 2006).

Table 3.

EET and 20-HETE receptors

GPR75	Parameter measured	Ki (nM)								References
Receptor/Expression		20-HETE
hGPR75/HEMa	disp	0.1								Garcia et al. (2017)
EC50/IC50 (nM)
hGPR75/CHO-K1b	Ca2+↑	0.12								Ignatov et al. (2006)
hGPR75/CHO-K1c	IP3↑	0.3								Ignatov et al. (2006)

FFAR1	Parameter measured	Ki (nM)								References
Receptor/Expression		20-HETE	14,15-EET	11,12-EET	8,9-EET	AA	Linoleic	α-linolenic	Palmitic
hFFAR1/HEK293d	Disp	–	6400	2700	–	–	–	–	–	Park et al. (2018)
FFAR1/COSb	Disp	4.5	–	–	–	–	–	–	40	Tunaru et al. (2018)
EC50/IC50 (nM)
hFFAR1/CHOb	Ca2+↑	–	–	1,400	6100	2400	1800	2000	6800	Itoh et al. (2003)
hFFAR1/HEK293b	Ca2+↑	–	580	910	–	3900	–	–	–	Park et al. (2018)
FFAR1/COSb	Ca2+↑	6300	–	–	–	> 320,000	32,000	13,000	32,000	Tunaru et al. (2018)

TRPV1	Parameter measured	Ki (µM)								References
Receptor/Expression		20-HETE	12(S)-HPETE	15(S)-HPETE	5(S)-HETE	LTB4	Capsacin
rTRPV1/HEK293e	Disp	–	0.35	–	–	–	2.5			Shin et al. (2002)
EC50/IC50 (nM)
hTRPV1/HEK293e	Current	10	–	–	–	–	0.3			Wen et al. (2012)
hTRPV1/HEK293f	Current	–	8	8.7	9.2	11.7	–			Hwang et al. (2000)
hTRPV1/CHOe	Current	–	–	–	–	–	0.5			McIntyre et al. (2001)
hTRPV1/XOg	Current	–	–	–	–	–	2.2			Cortright et al. (2001)

Agonist parameters given for Human (h) and rat (r) receptors in the indicated cell line. Parameter measured: Ca²⁺↑, increase in [Ca²⁺]_i; Current, current response from whole-cell patch clamp; disp, displacement assay; IP₃↑, increase in intracellular IP₃. Cell type abbreviations: CHO, Chinese hamster ovary cells; CHO-K1, subclone from the parental CHO cell line; COS, African green monkey kidney cell line; HEK293, Human embryonic kidney 293 cells; HEM, Human Epidermal Melanocytes; XO, xenopus oocytes. Agonist abbreviations: 11,12-EET, 11,12-epoxy-5Z,8Z,14Z-eicosatrienoic acid; 12(S)-HPETE, 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 14,15-EET, 14,15-epoxy-5Z,8Z,11Z-eicosatrienoic acid; 15(S)-HPETE, 15S-Hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; 20-HETE, 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid; 5(S)-HETE, 5S-Hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 8,9-EET, 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid; AA, arachidonic acid LBT₄, leukotriene B₄. a) Estimated from graph for displacement of [³H]20-HETE and K_i calculated using the Cheng-Prusoff equation. b) Determined from Ca²⁺ mobilization. c) Determined from IP₃ production. d) Determined by displacement of agonist [³H]TAK-875. e) Peak current response from patch clamp of whole cells. f) Estimated from graph of voltage response measured with whole cell voltage clamping. g) Current response for hTRPV1 expressed in xenopus oocytes (XO) measured with inside-out patch activated single channel currents

Mechanism of cell activation

In human microvascular endothelial cells, 20-HETE signaling proceeds through a Gα_q11 mediated pathway, where the agonist-induced dissociation of Gα_q11 leads to an increase in intracellular IP₃ (likely PLC activated) which results in an increase in [Ca²⁺]_i, as expected for Gα_q signaling. The Gα_q11 dissociation also enhances GPCR-kinase interacting protein-1 (GIT1) which in turn leads to activation of the tyrosine-protein kinase c-Src which activates epidermal growth factor receptor (EGFR). Activation of EGFR precipitates an EGFR/MAPK/IKK/NFκB signaling pathway that induces angiotensin-converting enzyme (ACE) transcription, ultimately leading to the conversion of angiotensin I to the vasoconstrictor angiotensin II. This pathway also induces phosphorylation of a subunit (Maxi-Kβ) of the large-conductance Ca²⁺ channel BK_Ca (Garcia et al. 2017). This serves to deactivate the BK_Ca channel (Vetri et al. 2014), ultimately leading to membrane depolarization, increased [Ca²⁺]_i, and stimulation of the contractile apparatus of vascular smooth muscle. In contrast, 20-HETE acts in an anti-hypertensive role in the kidney by initiating sodium reabsorption (Zhang et al. 2018).

As would be expected, RANTES/CCL5 exhibits similar effects upon binding to GPR75. RANTES/CCL5 activation of GPR75 transfected into HEK293 cells stimulates a release of IP₃ in a PLC dependent manner that leads to an increase in [Ca²⁺]_i, consistent with a Gα_q signaling pathway (Ignatov et al. 2006). Enhanced phosphorylation (activation) of MAPK and Akt is also observed. This event also occurs in neuroblastoma cells that express GPR75 and no other RANTES/CCL5 receptor (Dedoni et al. 2018). In addition, GPR75 serves as a RANTES/CCL5 receptor in human β-cells where stimulation produces an increase in [Ca²⁺]_i that leads to short-term elevation of insulin at both sub-stimulatory and maximal stimulatory glucose concentrations (Tunaru, 2016). More recent studies confirm that CCL5 stimulation of GPR75 is effective at increasing insulin secretion by β-cells (Gençoğlu et al. 2019). Comparable studies for the effect of 20-HETE on β-cells has not been reported.

In contrast to these studies, one attempt to reproduce the elevation of [Ca²⁺]_i by either RANTES or 20-HETE failed, and the authors suggest that the aforementioned studies were actually monitoring the activity of an unknown receptor (Iyinikkel 2018). However, the radioligand displacement studies and radiolabeled binding to wild type and the absence of binding to a GPR75 knockdown clearly show that 20-HETE is a ligand for GPR75 (Garcia et al. 2017).

Regulation

Regulation by phosphorylation has not been reported to date. Several groups have reported that agonist induced β-arrestin binding does not occur with GPR75 and thus inhibition and downregulation do not follow this pathway (Garcia et al. 2017; Iyinikkel 2018; Southern et al. 2013). However, GPR75 does internalize into punctate vesicles and internalization occurs through a dynamin-dependent pathway (Iyinikkel 2018). Interestingly, internalization occurs in the absence of added agonist, suggesting constitutive activity or activation by a yet-to-be discovered agonist in the media.

FFAR1/GPR40

Introduction

The human free fatty acid receptor 1 (hFFAR1, previously designated as hGPR40) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is activated by the potent agonist 20-HETE, EETs (eicosatetraenoic acids), and free fatty acids derived from dietary triglycerides (see Itoh et al. 2003). FFAR1 plays a major role in glucose metabolism, whereby stimulation leads to the secretion of insulin and glucagon (Tunaru et al. 2018; Trauelsen et al. 2018).

Human FFAR1 (GPR40, FFAR1, UniProtKB-O14842) is translated as a 300 amino acid polypeptide with a calculated molecular weight of 31.5 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 8 residues and a cytosolic C-terminal with 21 residues. There are no additional isoforms and one coding SNP variant (R211H) reported with unknown biological effect (Gerhard et al. 2004). There are several reported X-ray structures, (e.g., PDB reference 4PHU, 5KW2). Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hFFAR1 is expressed in only a few tissues with highest expression in bone, brain, pancreas, ovary, and digestive system (Uhlén et al. 2015). It is well established that FFAR1 is a receptor for free long chain fatty acids (FFA) with at least 12 carbons as well as various EETs, all with EC₅₀ values in the low micromolar range (Fig. 1, Table 3) (Mohapatra and Nau 2005; Park et al. 2018; Kotarsky et al. 2003) and 20-HETE (Tunaru et al. 2018) with a similar concentration dependence. A recent report shows that FFAR1 is also activated by lysophosphatidyl choline (LPC) (Drzazga et al. 2018).

Mechanism of cell activation

Although the overall function for FFAR1 is tissue dependent, it clearly signals primarily through a Gα_q pathway and partially through Gα_i (Itoh et al. 2003; Briscoe et al. 2003), both resulting in an increase in [Ca²⁺]_i. The former proceeds through a lysolecithin-inositol trisphosphate (PLV/IP₃) pathway. The latter involves the activation of PLC by the Gβγ subunit released upon Gα_i activation, producing IP₃ that results in an increase in [Ca²⁺]_i while at the same time directly inhibiting adenylate cyclase, thus reducing intracellular cAMP (Nolan et al. 2006).

The primary function of FFAR1 in the pancreas and the digestive system is the enhancement of glucose-stimulated insulin secretion (GSIS) through stimulation of the respective enteroendocrine cells, albeit through vastly different mechanisms. It is well established that under conditions of elevated plasma glucose, pancreatic β-cells import glucose which serves to drive the synthesis of ATP. This in turn leads to depolarization of the plasma membrane and Ca²⁺ influx. Increased [Ca²⁺]_i stimulates fusion of insulin containing vesicles with the plasma membrane thus releasing insulin to the circulatory system. In addition to glucose transport proteins, pancreatic β-cells also express high amounts of FFAR1. Activation of this receptor with long chain fatty acids or their oxidized derivatives leads to a Gα_q-mediated activation of PLC which, through a PLC/IP₃ pathway, increases the release of Ca²⁺ from the ER, leading to an increase [Ca²⁺]_i that serves to enhance GSIS (Tunaru et al. 2018; Houthuijzen 2016). These activators are unlikely to come directly from dietary fatty acids, as the concentrations necessary to activate FFAR1 (Table 3) are well beyond what would be expected for free fatty acids in plasma. However, localized high concentrations of FFA could be facilitated by intracellular lipases activated by glucose that are in turn transported into the localized extracellular environment. Oxidized derivatives of FFAs can be readily produced by intracellular cytochromes and transported to the localized extracellular environment (Tunaru et al. 2018). The lower EC₅₀ for oxidized derivatives compared to FFAs make the oxidized derivatives the more likely physiological agonists (Tunaru et al. 2018; Itoh et al. 2003; Trauelsen et al. 2018; Park et al. 2018). In addition to β-cell expression, FFAR1 is also expressed in the endocrine cells of the gastrointestinal tract. Here, in response to activation by dietary FFAs or autocrine release of FFAs or oxidized derivatives, FFAR1 stimulates the release of incretins (GLP-1 and GIP) which serve to augment GSIS (Edfalk et al. 2008; Luo et al. 2012).

FFAR1 receptor stimulation is also known to enhance glucagon excretion from α-cells. At face value this seems counterintuitive to the known effects of 20-HETE on insulin secretion from β-cells. However, as Taruelson et al. (2018) explain, any 20-HETE stimulation of FFAR1 is likely to be circumvented by paracrine inhibition of α-cells by β-cell secretory products. However, at low glucose concentrations where insulin is low, circulating long chain fatty acids released from fat stores serve to stimulate FFAR1 to release glucagon.

FFAR1 also serves important functions in other tissues. For example, in bone this receptor exerts a protective effect by inhibiting osteoclast differentiation (Wauquier et al. 2013). In the brain it is involved in antinociceptive activity involving the descending pain control system (Aizawa et al. 2016; Mancini et al. 2015). This receptor has also been shown to be intimately involved in the mediation of ovarian cancer growth (Munkarah et al. 2016).

Regulation

To date, there are no reports describing desensitization of hFFAR1 by phosphorylation, including GRK phosphorylation, that would lead to possible arrestin binding and subsequent internalization. However, the FFAR1 agonist dicosahexaenoic acid (DHA) does cause desensitization in monkey bone marrow derived stromal cells (BMSC) through internalization of FFAR1 (Kaplamadzhiev et al. 2010). Murine FFAR1 expressed in HEK-293 cells does interact with both β-arrestin 1 and 2 (Mancini et al. 2015) when stimulated with the strong synthetic agonist TAK-875, supporting a possible β-arrestin-mediated internalization event. However, hFFAR1 expressed in HeLa cells does not exhibit β-arresting binding (Williams-Bey et al. 2014) when exposed to DHA binding, suggesting some other mechanism for internalization in that system.

Regulation of FFAR1 at the genetic level has also been examined. Following treatment of clonally-expanded monkey bone marrow-derived stromal cells (BMSC) with basic fibroblast growth factor (βFGF), the expression of FFAR1 mRNA and protein increases significantly (Kaplamadzhiev et al. 2010).

TRPV1

Introduction

The human transient receptor potential cation channel subfamily V member 1 receptor (hTRPV1) is a member of the transient receptor potential Ca(2 +) channel (trp-cc) family. It is activated by various oxidized derivatives of both AA and linoleic acid, and various vanilloids and endocannabinoids. TRPV1 functions as a general sensor for noxious stimuli including heat, acid, proinflammatory stimulants, and painful chemical stimulants (review, Szallasi and Blumberg 1999). TRPV1 is a non-selective cation channel with high permeability for divalent cations (Caterina et al. 1997).

hTRPV1 receptor (VR1, Capsaicin receptor, OTRPC1, UniProtKB-Q8NER1) is translated as a 540 amino acid polypeptide with a calculated molecular weight of 95.0 kDa. There is a second isoform (isoform 2, UnitrotKB-Q8NER1-3) that differs in sequence for the first 150 amino acids and four potential isoforms that are computationally mapped (UniProt E7EQ78, I3L1R6, E7ESJ2, and A0A3B3ISI9). Lu et al. (2005) reported a third isoform (UniProtKB—Q52PU4) identical to the canonical isoform with the exception of a missing 60 amino acid corresponding to the loss of exon 7 and dubbed it hTRPV1b in reference to the similarly cleaved rat isoform. Sequence analysis reveals that hTRPV1b and the computationally mapped E7ESJ2 are one in the same. There are 11 known coding SNP variants for hTRPV1: P91S, M315I, T469I, T505A (Cortright et al. 2001), I585V (Hayes et al. 2000), T219A, V288G, V458M, F589L, T612M, and D625N for the canonical isoform with unknown biological impact (Landrum et al. 2016). Coding SNP variants for hTRPV1b or isoform 2 have not been reported to date beyond notation in the Ensembl database. There are no complete X-ray structures reported for any isoform but there is one partial structure for the N-terminus of hTRPV1 (residue 101–365, PDB entry 6L93). There are a number of nearly complete structures reported for rat TRPV1 (rTRPV1) determined using cryo-electron microscopy (e.g., PDB entry 5IRX, residues 110–764, 2.95 Å resolution). The fact that the sequence homology between rTRPV1 and hTRPV1, hTRPV1b and isoform 2 are 86%, 62% and 90% respectively make the rTRVP1 structure a good working model for each of these isoforms. The overall structure is unique among those discussed here in that there are only six transmembrane helices and both of the substantial N- (433 residues) and C- termini (152 residues) are cytosolic (Schumacher and Eilers, 2010).

Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally. However, glycosylation of N604 on rTRPV1 has been confirmed (Rosenbaum et al. 2002; Jahnel et al. 2001; Veldhuis et al. 2012) but predicted probabilities for glycosylation at this site are low for both the human (hTRPV1-1; N604) and rat proteins. Although phosphorylation of hTRPV1 has yet to be reported, the 85.8% homologous rat TRPV1 has been shown to be phosphorylated at S502 (Numazaki et al. 2002; Bhave et al. 2003; Jung et al. 2004), S800 (Numazaki et al. 2002; Bhave et al. 2003), T704 (Jung et al. 2004), S6, T144, T370 and S116 (Bhave et al. 2002), corresponding to S502, S801, T705, S6, T146, T370 and S117 on hTRPV1.

Expression and characterization

hTRPV1 is widely expressed in most tissues with higher expression in female specific tissues, skin, and the digestive tract (Uhlén et al. 2015; Montell 2005). Although widely expressed, nociceptors within these tissues exhibit the highest expression of hTRPV1, transducing a localized response to noxious stimuli (Mandadi and Roufogalis 2008; Ständer et al. 2004). As noted above, TRPV1 is a non-selective cation channel with a preference for divalent cations. The relative permeability of rTRPV1 for cations is Ca²⁺  > Mg²⁺  > Na⁺ ≈ K⁺ ≈ Cs⁺ where permeability ratios were determined to be: P_Ca/P_Na = 9.6 and P_Mg/P_Na = 4.99 (Caterina et al. 1997). The expression and function of TRPV1 in nervous tissue is particularly interesting. In human brain tissue hTRPV1 is expressed in all regions with slightly higher amounts in the cerebellum, midbrain, and hypothalamus (Uhlén et al. 2015). Specifically, TRPV1 is primarily expressed in nociceptors (Caterina and Julius 2001) and serves as a sensor for heat, acid, proinflammatory stimulants, as well as noxious xenobiotics such as capsaicin, the spicy hot compound found in chili peppers. hTRPV1b is also expressed in many tissues with particularly high expression in the cerebellum, fetal brain and dorsal root ganglia (Vos et al. 2006). Tissue expression of isoform 2 has not been reported, but it is likely that it too is expressed in neurons. The deletion of exon 7 in TRPV1b is reported to alter the sensitivity to stimulants but this effect is controversial. Lu et al. (2005) indicate that this isoform only responds to heat and not to capsacin or protons as observed for TRPV1 whereas Vos et al. (2006) report that this receptor does not respond to any the stimulants found for TRPV1. Specific agonists for isoform 2 have not been reported.

In vivo, the rTPRV1 receptor is found in oligomers with tetramers as the most prevalent (Kedei et al. 2001). Oligomerization is promoted by the presence of agonist but blocked in the presence of the antagonist capsazepine. Another report reveals that very strongly adhered dimers can survive SDS-PAGE analysis, but only if residue N604 is glycosylated and that the N604S mutant rat TRPV1 only forms monomers, confirming the importance of N604 glycosylation (Rosenbaum et al. 2002). hTRPV1 and hTRPV1b also form multimeric complexes of dimers, trimers and tetramers depending on conditions with tetramers preferred by TRPV1b (Vos et al. 2006). Oligomerization of isoform 2 has not been reported.

The TRPV1 receptor binds to and is activated by several oxidized derivatives of AA (5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 20(S)-HETE, and LTB₄) all with similar EC₅₀ values around 10 nM (Fig. 1, Table 3). TRPV1 also binds a number of different oxidized derivatives of linoleic acid (e.g., 9(S)-HODE, ( ±) 13-HODE, and 15(S)-HAEA) with EC₅₀ values in the micromolar range. Further, it binds to and is activated by a variety of cannabinoids with sub-micromolar affinity (De Petrocellis et al. 2000) as well as the non-selective opioid antagonist naloxone (Melkes et al. 2020). It is also a high affinity receptor for the plant-derived vanilloid capsaicin (EC₅₀ = 0.3–2.2 nM), the original namesake for this receptor. These and additional PUFA, N-acyl derivative, and other agonists are discussed in detail in a recent review by Benítez-Angeles et al. (2020). Activation of TRPV1b or isoform 2 by any oxidized PUFA has not been reported.

TRPV1 is also gated by both heat and pH. hTRPV1 gates at approximately 44 °C when expressed in either xenopus oocytes (Cortright et al. 2001) or HEK293 cells (Hayes et al. 2000) and ion currents increase significantly with a moderate increase in temperature. hTRPV1 gates with decreasing pH that begins around pH 6.5 with a pK of 5.4–5.5 (Hayes et al. 2000; McIntyre et al. 2001). Lower pH also sensitizes hTRPV1 to the agonist capsaicin (Cortright et al. 2001). The effect of the potent antagonist capsaizepine on pH gating is controversial. One report indicates that capsaizepine inhibits 90% of the pH-induced ion current at pH 5 (Cortright et al. 2001), whereas another report indicates that no such effect is observed (McIntyre et al. 2001). Protons also reduce the temperature threshold for gating, even at moderately acidic pH (≤ 5.9) (Tominaga et al. 1998). Although there appears to be some synergy in signaling, whether the signaling mechanism for protons, heat or agonist share commonalities remains to be determined.

Mechanism of cell activation

Stimulation of neural cells expressing TRPV1 with an agonist such as capsaicin leads to a rapid influx of cations, particularly Ca²⁺ and Na⁺ resulting in depolarization of the membrane (Marsh et al. 1987) and the subsequent release of neurotransmitters and neuromodulators from the nociceptor (Caterina and Julius 2001). The depolarization appears to be bimodal with time constants of 6.7 and 52 ms for rTRPV1 when expressed in HEK293 cells and monomodal with a time constant of 385 ms in native dorsal root ganglion (DRG) neurons. The large difference in depolarization rates may be due to the presence of factors found only in native systems (Gunthorpe et al. 2000). The presence of oligomers in native systems could very well be one of these mitigating factors.

Regulation

Regulation by phosphorylation is a common theme for many receptors. Treatment of rat DRG or HEK293 cells expressing hTRPV1 with phorbol 12-myristate-13-acetate (PMA), an activator of PKC, leads to an average of two-fold enhancement of ion current through TRPV1 when in the presence of capsaicin at less than maximal activation concentrations, but not at maximal concentrations (Vellani et al. 2001). Enhancement of both pH- and heat-induced ion current by PKC activation is also observed. However, PMA does not produce significant ion current in most DRGs or HEK293 cells stably transfected with hTRPV1 in the absence of agonist. These and later results (Bhave et al. 2003) suggest that PKC does not actuate the channel but enhances the action of other stimuli. Similar results are also obtained for rTRPV1 expressed in Xenopus oocytes upon activation of PKC (Premkumar and Ahern 2000). In contrast, however, the ion current in the absence of capsaicin or anandamide is more pronounced. The effect of PMA on rTPRV1 and Ser and Thr to Ala mutants expressed in HEK293 cells reveals that S502 and S800 are the sites of PKC phosphorylation and that each contributes to the total augmentation of capsaicin-induced ion current and reduction of the threshold temperature for heat-induced ion-current (Numazaki et al. 2002).

Phosphorylation of TRPV1 by cAMP-dependent PKA also enhances agonist-, heat- or pH-induced ion current. Exposure of rat nociceptive neurons or HEK293 cells transfected with rTRPV1 to the cAMP activator forskolin potentiates heat-induced ion current and exposure to the PKA inhibitor PKI_14-22 prevents potentiation (Rathee et al. 2002). The experiment repeated with mutants T144D, T370D and S502D shows that phosphorylation at each of these sites potentiates heat-induced ion current with phosphorylation at S502 rendering the greatest sensitization. Later work reveals that PKA-dependent phosphorylation of S116 also potentiates capsaicin-induced ion-current (Bhave et al. 2002). Further, sensitization of the rTRPV1 response to both capsaicin and heat by PKC, especially PKCε, involves phosphorylation at S502 or S800 (Numazaki et al. 2002) and this sensitization is not modified in the calcineurin-mediated desensitization (Mohapatra and Nau 2005).

The response of rTRPV1 to capsaicin-induced ion current in both cultured rat neurons and HEK293 cells transfected with rTRPV1 is enhanced through phosphorylation by Ca²⁺-calmodulin dependent kinase II (CaMKII) (Jung et al. 2004). Data obtained from site specific mutants indicates that phosphorylation at T704 allows for activation but is refractory to sensitization by other kinases (e.g., PKA and PKC) unless S502 is also phosphorylated. Further work reveals that dephosphorylation by the calmodulin-dependent phosphatase calcineurin reverses activation by reducing the ability of rTRPV1 to bind agonist. Hence, the regulation of rTRPV1 is controlled by a balance between calcium-mediated phosphorylation by CaMKII and calcium-mediated dephosphorylation by calcineurin with augmentation of activity by PKA and PKC. The apparent calcium-linked paradox has yet to be explained and may be quite complex; several mechanisms have been proposed (Jung et al. 2004). Interestingly, lack of phosphorylation at either S502 or T704 does not affect the response of rTRPV1 to acid, indicating that agonist-induced and acid-induced sensitization proceed through different pathways (Jung et al. 2004).

Both desensitization and ion selectivity but not expression are functions of the glycosylation state of N604 (Veldhuis et al. 2012). Capsaicin-induced ion fluxes are sustained for wild type rTRPV1 expressed in HEK293 cells whereas mutant (N604T), non-glycosylated rTRPV1 is rapidly desensitized. Both forms of rTRPV1 are equally expressed on the plasma membrane surface and thus the glycosylation state does not control surface expression. Capsaicin activation of the wild type channel exhibits a concentration dependent influx of the di-cationic dye YO-PRO-1, whereas the mutant shows no such influx, thus glycosylation controls the selectivity of the receptor. These results leave open the possibility that maintenance of the glycosylation state could be used for the regulation of this channel in vivo (Veldhuis et al. 2012).

When expressed together, TRPV1b associates with TRPV1 in heteromeric complexes. The net result is a negatively regulatory effect on TRPV1 (Vos et al. 2006; Lu et al. 2005). Thus, TRPV1b may be a regulator of TRPV1 activity in vivo.

Leukotriene receptors

Introduction

Leukotrienes represent a specific class of oxidized derivatives of AA containing three conjugated and one unconjugated double bond of which several are derivatized with glutathione that may be proteolyzed to form different leukotriene products. The synthesis begins with the peroxidation of AA by polyunsaturated fatty acid 5-lipoxygenase (ALOX5) to the highly unstable leukotriene A₄ (LTA₄) (Fig. 1). LTA₄ is readily converted to leukotriene B₄ through a step-wise hydrolysis by leukotriene A-4 hydrolase (LTA4H) or conjugated with glutathione by leukotriene C₄ synthase (LTC4S) to form leukotriene C₄ (LTC₄). Stepwise proteolysis by glutathione hydrolase 1 proenzyme (GGT1) and then dipeptidase 1 (DPEP) of the glutathione attached to LTC₄ produces leukotriene D₄ (LTD₄) and leukotriene E₄ (LTE₄) respectively. LTB₄ is a potent chemotaxis stimulating molecule serving to recruit and activate eosinophils, monocytes, and neutrophils (Haeggström 2000; Samuelsson 1983; Crooks and Stockley 1998). The cysteinyl-leukotrienes, LTC₄, LTD₄ and LTE₄, as they are known are potent bronchoconstrictors, serving to increase vascular permeability (Samuelsson 1983).

LTB4R

Introduction

Human leukotriene B₄ receptor 1 (hLTB4R) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is strongly activated by leukotriene B₄ (LTB₄), a potent inflammatory chemoattractant. Stimulation of this receptor leads to signaling through MAPK and PI3K pathways (Tong et al. 2005; Lindsay et al. 1998). LTB4R also has a role in enhancing allergic airway inflammation, inflammatory arthritis and atherosclerosis (Yokomizo et al. 2018).

hLTB4R (BLT1, LTB4-R1, P2Y7, UniProtKB-Q15722) is translated as a 352 amino acid polypeptide with a calculated molecular weight of 37.6 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 19 residues and a long cytosolic C-terminal with 63 residues. There are no additional isoforms and two coding SNP variants reported: L346F (Stelzer et al. 2016) and A79S (Landrum et al. 2016). The biological ramifications of these SNPs are unknown. There are no reported X-ray structures. There is one model available in the SWISS-Model repository using Leukotriene B₄ receptor BLT1 in complex with BIIL260 (72.8% sequence homology; PDB entry 5X33) that may serve as a working model (Waterhouse et al. 2018). Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, only some of these have been confirmed experimentally. Mutation of Ser and Thr residues to Ala abolishes agonist-induced internalization, suggesting that the predicted phosphorylation at T308, S310, S313, S314 and T315 are potential sites for phosphorylation (Aratake et al. 2012). Phos-tag gel electrophoresis studies confirm these phosphorylation sites and two more, S320 and T324, where phosphorylation of S310 and T308 are LTB₄ induced and the others represent basal phosphorylation (Nakanishi et al. 2018).

Both Resolvin E¹ and Resolvin E² (see below), oxidized derivatives of docosahexaenoic acid, serve as equally potent partial agonists for the LTB4R receptor and thus compete with LTB₄ but activate the receptor to a lesser extent than LTB₄ (Oh et al. 2012). The primary receptor for these resolvins is CMKLR1 (see below).

Expression and characterization

hLTB4R is primarily expressed in the bone, lymphoid tissue and blood, but is also found in other tissues in lower amounts, particularly in the esophagus and digestive system (Uhlén et al. 2015). LTB4R is the high affinity receptor for leukotriene B₄ (LTB₄) with a K_i in the sub-nanomolar range, but also exhibits strong binding to 12(R)-HETE (K_i = 30 nM) (Fig. 1, Table 2) (Yokomizo et al. 1997). EC₅₀ values for LTB₄ are in the 3–10 mM range (Yokomizo et al. 1997). EC₅₀ values for 12(R)-HETE have yet to be reported.

Mechanism of cell activation

LTB4R is yet another receptor with G-protein promiscuity, depending on the G-protein availability or the cell type in which it is expressed. LTB4R couples mainly with Gα_i-like proteins, but also with Gα_q and Gα₁₆, leading to a reduction in cAMP facilitated by the former and a PLC/IP₃-mediated increase in [Ca²⁺]_i by the latter two (Saeki and Yokomizo 2017; Malfacini et al. 2019; Kuniyeda et al. 2007).

The primary function for LTB4R is its involvement in the immune response which is characterized largely by its stimulation of leukocyte chemotaxis. Activation by LTB₄ induces neutrophil swarming (Lämmermann et al. 2013) and vascular smooth muscle cell migration (Bäck et al. 2005) to sites of tissue damage. The former is particularly important in allergic skin inflammation where the scratching response to irritants causes microinjuries that lead to neutrophil infiltration (Sadik et al. 2013). LTB4R activation is also involved in the recruitment of CD4⁺ and CD8⁺ T cells to sites of inflammation (Goodarzi et al. 2003; Tager et al. 2003).

Regulation

Ligand activated phosphorylation of LTB4R by either PKC, GRK2, GRK5 or GRK6 results in desensitization. T308 is the major but not the only site for GRK6 phosphorylation. However, only phosphorylation at T308 is associated with desensitization (Gaudreau et al. 2002). Nakanishi et al. (2018) expand these observations and delineate the cellular responses. They show that basal phosphorylation of LTB4R occurs in variable patterns in the absence of LTB₄. At low concentrations of LTB₄ the receptor adopts a high affinity configuration that stimulates the associated Gα_i to exchange out GDP for GTP, initiating chemotaxis. Following deactivation of Gα_i by GTP hydrolysis, S310 is phosphorylated which initiates further basal phosphorylation, producing a low affinity form. As migration up the LTB₄ concentration gradient continues, the higher LTB₄ concentrations stimulate phosphorylation at T308, a signal for further phosphorylation leading to additional responses including degranulation.

Desensitization through internalization is a common feature for GPCRs. Desensitization of LTB4R by ligand-induced, phosphorylation-triggered internalization has been observed. The cytoplasmic C-terminus is likely phosphorylated at residues T308, S310, S313, S314, and T315 and phosphorylation is required for ligand-induced internalization. Specific amounts of required phosphorylation and the importance of phosphorylation of specific residues is not known. The dileucine motif in the C-terminal helix (helix 8) is important in internalization and mutation of these leucines (L304 and L305) to alanines result in rapid ligand-induced internalization, suggesting that this motif inhibits receptor phosphorylation (Aratake et al. 2012). Interestingly, phosphorylation of LTB4R is not required for β-arrestin binding and subsequent β-arrestin-mediated internalization (Jala et al. 2005). Taken together, these results show that there are multiple pathways for LTB4R internalization.

Regulation of LTB4R at the genetic level has also been reported. Upregulation of LTB4R is accomplished through a Iκ kinase β/NF-κB-dependent pathway and upregulation can be induced by treatment with either IL-1β or immune-stimulating lipopolysaccharides (LPS) (Bäck et al. 2005).

Cysteinyl Leukotriene receptor 1 (CYSLTR1)

Introduction

Human cysteinyl leukotriene receptor 1 (hCYSLTR1) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family and a structural homolog of purigenic receptors (P2Y). It binds to and is activated by leukotrienes in the following order of preference leukotriene D₄ (LTD₄) >  > leukotriene E₄ (LTE₄) ≈ leukotriene C4 (LTC₄) >  > leukotriene B₄ (LTB₄) (Lynch et al. 1999). Stimulation of this receptor leads to signaling through a PLC/PI₃ pathway that leads to the elevation in [Ca²⁺]_i (Crooke et al. 1989; Watanabe et al. 1990; Ohshima et al. 2002; Yan et al. 2011; Sarau et al. 1999) and phosphorylation/activation of ERK/MAPK and various tyrosine kinases (Jiang et al. 2007; Boehmler et al. 2009). CYSLTR1 plays a major role in inflammation and is implicated in a number of diseases including atropic dermatitis, allergic rhinitis, asthma, and cardiovascular disease (review Yokomizo et al. 2018).

hCYSLTR1 (LTD₄ receptor, HG55, HMTMF81, UniProtKB- Q9Y271) is translated as a 337 amino acid polypeptide with a calculated molecular weight of 38.5 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 28 residues and a cytosolic C-terminal with 40 residues. There are no additional isoforms and one coding SNP variant reported (G300S) with unknown biological import (Thompson et al. 2016). There is also one N23 frameshift mutant found in malignant prostate tumors with otherwise unknown significance (Landrum et al. 2016). There are two X-ray structures available (PDB entry 6RZ4 and 6RZY). Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, some of which have been confirmed experimentally. Tyr phosphorylations are predicted for Y30 and Y319 but only Tyr phosphorylation without mention of a specific site has been reported (Naik et al. 2005). Ser phosphorylation by PKC on the C-terminal cytoplasmic tail (S313, S315, or S316) is confirmed (Naik et al. 2005).

Expression and characterization

hCYSLTR1 is primarily expressed in the bone, lymphoid tissue and blood, but is also found in other tissues in lower amounts, particularly in the brain and digestive system (Uhlén et al. 2015). CYSLTR1 is the high affinity receptor for leukotriene D₄ (LTD₄) with a binding IC₅₀ in the sub-nanomolar range and weaker binding (IC₅₀ in the 300–400 nM range) to LTC₄ and LTE₄ (Table 4) (Lynch et al. 1999). EC₅₀ values for LTD₄ are in the low nM range while others are significantly higher (Fig. 1, Table 4).

Table 4.

Leukotriene and Lipoxin receptors

CYSLTR1&2	Parameter measured	Ki (nM)									References
Receptor/Expression		LTB4	LTC4	LTD4	LTE4
hCYSLTR1/HLPM higha	Disp	–	7.9	7.7	89						Capra et al. (1998)
hCYSLTR1/HLPM lowa	Disp	–	96	46	15						Capra et al. (1998)
hCYSLTR1/COS-7b	Disp	–	–	25	–						Dupré et al. (2004)
EC50/IC50 (nM)
hCYSLTR1/Th2c	Ca2+↑	–	8	0.8	6						Parmentier et al. (2012)
hCYSLTR1/HEK293c	Ca2+↑	–	1,483	11.6	391						Yan et al. (2011)
hCYSLTR1/HEK293c	Ca2+↑	–	24	3	240						Sarau et al. (1999)
hCYSLTR1/COS-7c	Ca2+↑	–	–	3.4	–						Carnini et al. (2011)
hCYSLTR2/HEK293Tc	Ca2+↑	–	11.2	0.9	83.2						Foster et al. (2016)
hCYSLTR2/HEK293c	Ca2+↑	–	95	145	1208						Yan et al. (2011)
hCYSLTR2/HEK293Tc	Ca2+↑	> 3000	67	104	2300						Heise et al. (2000)
hCYSLTR2/HEK293Tc	Ca2+↑	–	9	4.4	293						Nothacker et al. (2000)
hCYSLTR2/COS-7c	Ca2+↑	–	–	9	–						Carnini et al. (2011)
hCYSLTR2/HUVECd	Ca2+↑	–	–	–	–						Carnini et al. (2011)

GPR17	Parameter measured	EC50/IC50 (nM)									References
Receptor/Expression		LTB4	LTC4	LTD4	LTE4	UDP	UDP-Glc	UDP-Gal	MDL29,951
hGPR17-1/1321N1e	cAMP↓	–	0.33	7.2	–	1,140	12,000	1100	–		Ciana et al. (2006)
hGPR17-1/1321N1e	cAMP↓	–	65	5.9	–	4,600	530	–	–		Ciana et al. (2006)
hGPR17-1/COS-7e	cAMP↓	–	14.8	4.4	–	–	–	–	–		Ciana et al. (2006)
hGPR17-1/HEK293e	cAMP↓	–	–	–	–	1,060	9500	729	–		Ciana et al. (2006)
hGPR17-2/1321N1meme	cAMP↓	–	0.68	2.1	–	264	2390	295	–		Pugliese et al. (2009)
hGPR17/1321N1memf	disp	–	–	10	–	–	1600	–	–		Daniele et al. (2011)
mGPR17/1321N1meme	cAMP↓	–	0.74	0.63	0.31	–	55–88	68	–		Lecca et al. (2008)
mGPR17/Oli-neu cellsg	Ca2+↑	–	–	–	–	–	–	–	10,000		Simon et al. (2016)
mGPR17-1/Oli-neu cellsh	DMR↑	–	–	–	–	–	–	–	320		Simon et al. (2016)
mGPR17/Oli-neu cellsi	ERK-P↑	–	–	–	–	–	–	–	100		Simon et al. (2016)
mGPR17/Oli-neu cellsj	cAMP↓	–	–	–	–	–	–	–	200		Simon et al. (2016)
hGPR17-1/HEK293h	DMR↑	–	–	–	–	–	–	–	5000		Hennen et al. (2013)
hGPR17-2/HEK293h	DMR↑	–	–	–	–	–	–	–	320		Hennen et al. (2013)
hGPR17-2/HEK293k	cAMP↓	–	–	–	–	–	–	–	6/1000		Hennen et al. (2013)
hGPR17-2/HEK293l	GTPγS	–	–	–	–	–	–	–	500		Hennen et al. (2013)
hGPR17-2/HEK293m	IP↑	–	–	–	–	–	–	–	320		Hennen et al. (2013)
hGPR17-2/HEK293n	Ca2+↑	–	–	–	–	–	–	–	60		Hennen et al. (2013)

P2Y12	Parameter measured	EC50/IC50 (nM)									References
Receptor/Expression					LTE4	ADP	2MeSADP	PRPP
hP2Y12-G16alpha/CHOc	Ca2+↑				1.3	–	0.059	7.8			Nonaka et al. (2005)
hP2Y12/purifiedn	GTPhyd↑				–	30,000	16	–			Bodor et al. (2003)
hP2Y12/COS-7o	IP↑				–	2400	0.6	–			Bodor et al. (2003)
hP2Y12/1321N1e	cAMP↓				–	6.8	0.07	–			Kauffenstein et al. (2004)
hP2Y12/CHOp	Akt-P↑				–	–	0.7	–			Soulet et al. (2004)
hP2Y12/CHOq	MAPK-P↑				–	–	0.6	–			Soulet et al. (2004)

FPR2/ALX	Parameter measured	Ki (nM)									References
Receptor/Expression		RvD1	LTC4	LTD4	LXA4	LXD4	ATLa1	ATLa2	15-deoxy-LXA4	15-epi-LXA4
hFPR2/HEK293f	Disp	–	–	–	0.02	–	0.03	0.03	100	–	Chiang et al. (2000)
hFPR2/PMNf	Disp	–	39	35	0.94	–	–	–	–	–	Chiang et al. (2000)
hFPR2/CHOf	Disp	–	–	–	5.6	79.9	–	–	–	–	Fiore et al. (1994)
hFPR2/hum leukocyter	RvD1	0.17	–	–	–	–	–	–	–	–	Krishnamoorthy et al. (2010)
mFPR2/CHOk	Disp	–	–	–	2	–	–	–	–	–	Takano et al. (1997)
EC50/IC50 (nM)
hFPR2/THP-1 s	laminin	–	–	–	0.8	–	0.08	0.08	–	–	Maddox et al. (1997)
hFPR2/HEK293t	β-arrestin	–	–	–	–	–	0.0029	–	–	0.0029	Sun et al. (2009)
hFPR2/HEK293t	β-arrestin	0.0012	–	–	0.0011	–	–	–	–	–	Krishnamoorthy et al. (2010)
hFPR2/HEK293t	β-arrestin	0.045	–	–	–	–	–	–	–	–	Krishnamoorthy et al. (2012)
hFPR2/HEK293e	cAMP↓	–	–	–	–	–	–	–	–	0.5	Ge et al. (2020)
hFPR2/HEK293c	Ca2+	–	–	–	–	–	–	–	–	< 10 μM	Ge et al. (2020)

OXGR1	Parameter measured	Ki (nM)									References
Receptor/Expression				LTD4	α-KG	AMP	Adenosine
mOXGR1/HEK293u	disp			6	–	3	–				Kanaoka et al. (2013)
mOXGR1/CHOu	disp			2	–	–	–				Kanaoka et al. (2013)
EC50/IC50 (nM)
hOXGR1/HEK293v	Ca2+↑			–	69,000/32,000	–	–				He et al. (2004)
hOXGR1/eosinophilsw	cAMP↑			≤ 10	–	–	–				Steinke et al. (2014)
hOXGR1/HEK293c	Ca2+↑			–	–	920	670				Inbe et al. (2004)

Agonist parameters given for Human (h) and murine (m) receptors in the indicated cell line or membrane system. Parameter measured: akt-P↑; increae in akt phosphorylation; β-arrestin, β-arrestin binding; Ca²⁺↑, increase in calcium; cAMP↓, decrease in cAMP; cAMP↑ increase in cAMP accumulation; disp, displacement assay; DMR↑; increase in dynamic mass redistribution; ERK-P↑, increase in ERK phosphorylation; GTPhyd↑; increase in GTP hydrolysis rate; IP↑; increase in inositol phosphate production; laminin, laminin binding; MAPK-P↑; increase in MAP kinase phosphorylation; RvD1, resolvin D1 binding. Cell type abbreviations: CHO, Chinese hamster ovary cells; COS-7, African green monkey kidney cell line; HEK293, Human embryonic kidney 293 cells; HLPM, Human lung parenchyma membranes; hP2Y12-G16alpha, PPY2-G16alpha fusion protein; HUVEC, Human umbilical vein endothelial cells; Oli-neu, murine oligodendroglial precursor cells; PMN, human polymorphonuclear leukocytes; Th2, T helper 2 cells; THP-1, Human monocyte derived from patient with acute monocytic leukemia. Agonist abbreviations: 2MeSADP, 2-methylthio-adenosine-5'-diphosphate; ALTa1, 15(R/S)-methyl-LXA₄, a stable LXA₄ (15S-LXA₄) analog; ALTLa2, 15-epi-16-(para-fluoro)-phenoxy-LXA₄ a stable 15R-LXA₄ analog; AMP, adenosine monophosphate; α-KG, α-ketoglutarate; LTB₄, leukotriene B₄; LTC₄, leukotriene C₄; LTD₄, leukotriene D₄; LTE₄, leukotriene E₄; LXA₄, lipoxin A₄; LXD₄, lipoxin D₄; MDL29,951, 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid; PRPP, 5-phosphoribosyl 1-pyrophosphate; RvD1, resolvin D1. a) Determined by displacement of agonist [³H]LTC₄ for LTD₄ determination and [³H]LTD₄ for LTC₄ and LTE₄ determination for high/low binding sites in human lung parenchyma membranes. b) Determined by displacement of agonist [³H]LTD₄. c) Determined from changes in intracellular Ca²⁺ d) Determined from changes in intracellular Ca²⁺ in human umbilical vein endothelial cells. e) Inhibition of forskolin-stimulated cAMP production. f) Estimated from graph for displacement of [11,12-³H]-LXA₄ and K_i calculated using the Cheng-Prussoff equation. g) Estimated from plot of %Ca²⁺ release versus agonist concentration. h) Estimated from plot of dynamic mass redistribution (DMR) assay versus agonist concentration. i) Estimated from a plot of ERK 1/2 phosphorylation versus agonist concentration. e) Estimated from a plot of cAMP versus agonist concentration. j) Estimated from a plot of cAMP versus agonist concentration as diminishment/enhancement. k) Estimated from a plot of [³⁵S]GTPγS binding versus agonist concentration. l) Estimated from a plot of total intracellular inositol phosphate accumulation versus agonist concentration. m) Estimated from a plot of total [Ca²⁺]_i accumulation versus agonist concentration. n) Purified hP2Y12 inserted into synthetic vesicles as measured by GTP hydrolysis. o) Inositol phosphate production. p) Akt phosphorylation. q) MAP-kinase phosphorylation. r) [³H]RvD1 binding. s) Adherence of human monocytic cell line THP-1 cell adherence to laminin. t) Binding of β-arrestins. u) Estimated from graph for displacement of [³H]LTE₄ and K_i calculated using the Cheng-Prussoff equation. v) [Ca²⁺]_i increase by aqueorin assay/FLIPER assay w) Increase in cAMP production

Mechanism of cell activation

CYSLTR1 signals through several different G-proteins. For example, in the differentiated promonocytic leukemia cell line U937, CYSLTR1 signals simultaneously through multiple pathways including Gα_q11, Gα_io, Gβγ subunits, as well as isoprenylated proteins such as members of the Ras family (Capra et al. 2003), resulting in a complex response leading to increased [Ca²⁺]_i (Pollock and Creba 1990). Similarly, human TH2 cell CYSLTR1 signals through both Gα_q and Gα_i (Parmentier et al. 2012), leading to an increase in [Ca²⁺]_i and a decrease in intracellular cAMP. In human epithelial cells signaling is known to occur through Gα_i3, stimulating an increase in [Ca²⁺]_i (Adolfsson et al. 1996).

CYSLTR1 plays a significant role in stimulating leukocyte chemotaxis. Activation by LTD₄ or other potent agonists are known to enhance recruitment of human eosinophils (Ohshima et al. 2002), monocytes (Woszczek et al. 2008), and TH2 cells (Parmentier et al. 2012). In a complementary fashion, CYSLTR1 also enhances vascular permeability, which in turn promotes leukocyte infiltration (Maekawa et al. 2002; Bochnowicz and Underwood 1995). This receptor is also known to control LTD₄ and LTC₄ induced vasoconstriction of human saphenous veins (Mechiche et al. 2004). Interestingly, in a mouse model for chronic pulmonary inflammation and fibrosis, CYSLTR1 exhibits an anti-inflammatory role by reducing the magnitude of septal thickening and deposition of reticular fibers (Beller et al. 2004). These results suggest a role for this receptor in the resolution of inflammation in lung tissue.

Regulation

Agonist-induced receptor internalization is a common theme for eicosanoid receptors and quite applicable to CYSLTR1. Stimulation of non-transformed human intestinal endothelial cells (Int 407 cells) with LTD₄ but not LTC₄ induces tyrosine phosphorylation of this receptor, ultimately leading to a rapid (5 min) internalization and localization to the nucleus. It recycles back to the plasma membrane when LTD₄ is removed. Internalization proceeds by clatherin-coated pits and trafficking of the early endosomes containing the receptor is facilitated by both the Ras-related protein Rab-5 and arrestin-3. Further, CYSLTR1 heterodimerizes with CYSLTR2 on the plasma membrane under basal conditions and LTD₄ stimulates internalization of CYSLTR1, leaving CYSLTR2 on the plasma membrane. Stimulation of the heterodimers with LTC₄ does not result in phosphorylation but does initiate internalization of both receptors without localization to the nucleus. Removal of LTC₄ reverses the process (Parhamifar et al. 2010). In contrast, CYSLTR1 can internalize by an arrestin-free internalization process where phosphorylation of S313, S315, or S316, or possible a combination by PKC inhibits internalization (Naik et al. 2005).

Heterodimers in human mast cells that naturally express both receptors modify the receptor response. When mast cells are treated with the shRNA-mediated knockdown of CYSLTR1, the response to LTD₄ is abrogated, indicating that CYSLTR1 is the primary receptor for LTD₄. Knockdown of CYSLTR2, on the other hand, doubles the response to LTD₄ by doubling the surface expression of CYSLTR1, presumably as dimers. Clearly, the heterodimer serves to regulate the cellular response to Cys-leukotrienes by regulating the amount of CYSLTR1 homodimer on the plasma membrane (Jiang et al. 2007). CYSLTR2 is not the only moderating receptor for CYSLTR1. The G-protein coupled receptor GPR17 heterodimerizes with CYSLTR1 and abolishes LTC₄- and LTD₄-mediated response (e.g. [Ca²⁺]_i). GPR17 expression not only mediates the surface expression of CYSLTR1 but also suppresses LTD₄ binding (Maekawa et al. 2009). CYSLTR1 and the generic purinergic G-protein coupled receptors (P2Y), of which GPR17 is a member, also interact. Activation of P2Y receptors with ATP or UDP induces desensitization of CYSLTR1 to LTD₄ but does not stimulate internalization. Conversely, stimulation of CYSLTR1 with LTD₄ has no effect on P2Y receptor responses and does initiate CYSLTR1 internalization. Apparently, crosstalk between G-proteins is intimately involved in regulation of responses at sites of inflammation (Capra et al. 2005).

Regulation of hCYSLTR1 at the transcriptional, translational, and posttranslational modification levels have been reported. Treatment of human bronchial smooth muscle cells (BSMC) with IL-13, transforming growth factor β (TGFβ) or interferon γ (IFNγ) upregulate the expression of CYCLTR1 but only IL-13 and IFNγ increase the amounts of CYSLTR1 mRNA. Evidently, the upregulation by TGF-β is either a translational or posttranslational event. Treatment of BSMCs with TGF-β or IL-13 increases their proliferation in response to LTD₄. These results indicate a synergy between cysteinyl leukotrienes (CysLTs) and specific cytokines in the expression of CYSLTR1 and BSMC proliferation (Espinosa et al. 2003). Others have reported that the IL-13 upregulation of hCYSLTR1 in human monocytes is biphasic in nature and IL-4 also increases CYSLTR1 mRNA levels 150-fold (Shirasaki et al. 2007).

Cysteinyl Leukotriene receptor 2 (CYSLTR2)

Introduction

Human cysteinyl leukotriene receptor 2 (hCYSLTR2) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is activated by leukotrienes in the following order of preference LTC₄ ≈ LTD₄ >  > LTE₄. Stimulation of this receptor in turn leads to signaling through a PLC/PI₃ pathway that leads to the elevation of [Ca²⁺]_i (Sarau et al. 1999; Moore et al. 2016; Carnini et al. 2011) and phosphorylation/activation of ERK/MAPK (Mehdawi et al. 2017; Mellor et al. 2003). CYSLTR2 plays a major role in inflammation (Mellor et al. 2003) and is implicated in several diseases including asthma (Thompson et al. 2016) and cancer (Moore et al. 2016; Brochu-Bourque et al. 2011).

hCYSLTR2 (CYSLT2, CYSLT2R, UniProtKB- Q9NS75) is translated as a 346 amino acid polypeptide with a calculated molecular weight of 39.6 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 42 residues and a cytosolic C-terminal with 39 residues. There are no additional isoforms and 8 reported coding SNP variants with unknown biological import: M201V (Thompson et al. 2016; Brochu-Bourque et al. 2011), E39Q, F50V, N103S, S236L, H242Q, L278I, R315K (Landrum et al. 2016). There are no X-ray structures available. There is one structural model available on the SWISS-MODEL website (PDB entry 6RZ6) (Waterhouse et al. 2018) to serve as a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hCYSLTR2 is primarily expressed in the brain, blood, and both male and female tissues, but is also found in other tissues in lower amounts, particularly in the muscle, bone, and the endocrine tissue Uhlén et al. 2015). CYSLTR2 is the high affinity receptor for both LTD₄ and LTC₄ with EC₅₀ values in the nM range (Fig. 1, Table 4).

Mechanism of cell activation

In human umbilical endothelial cells (HUVECs) and transfected HEK293 cells, hCYSLTR2 signals through Gα_q11, stimulating a PLC/IP₃ cascade leading to an increase in [Ca²⁺]_i and the formation of diacyl glycerol (DAG) (Sarau et al. 1999; Moore et al. 2016). One of the results of the increased [Ca²⁺]_i and DAG is the activation of PKC that in turn activates the MAPK pathway leading to enhanced phosphorylation of p65 (NF-κB family), ERK, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase (JNK) (Brochu-Bourque et al. 2010). Stimulation of hCYSLTR2 with agonist also results in significant transactivation and secretion of IL-8 (Mellor et al. 2003; Brochu-Bourque et al. 2011), a chemokine that induces chemotaxis of neutrophils and other granulocytes and stimulates phagocytosis. Interestingly, in human mast cells, IL-8 production is known to be mediated by the Gα_io family of G-proteins (Mellor et al. 2003). Secretion of IL-5, a known mediator in eosinophil activation and immunoglobulin secretion, is also enhanced, but this may involve CYSLTR1/ CYSLTR2 heterodimers.

Like CYSLTR1, CYSLTR2 also promotes vascular permeability, but contrary to CYSLTR1 it promotes significant vasodilation (Hui et al. 2004), an event in keeping with the regulatory role of CYSLTR2 in the CYSLTR1/CYSLTR2 heterodimer discussed above.

LTC₄ signaling through CYSLTR2 also promotes the synthesis of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) which catalyzes the oxidation of prostaglandin E₂ (PGE₂) to its inactive metabolite 15-keto-PGE₂ (Mehdawi et al. 2017). PGE₂ is well established as an important mediator in inflammation, driving acute inflammation, and a potent tumor promotor (Nakanishi and Rosenberg 2013). By mediating upregulation of 15-PGDH, CYSLTR1 is active in both anti-inflammatory and anti-tumor activity.

Regulation

Regulation of CYSLTR2 is reported to occur at both the surface expression and transcriptional levels. Priming of human mast cells (hMC) with IL-4 for 5 days results in a redistribution of CYSLTR2 to the plasma membrane, without changing the total cellular content of this receptor (Shirasaki et al. 2007; Mellor et al. 2003). Additionally, both LTD₄ stimulation and IL-13 upregulate CYSLTR2 mRNA levels (Shirasaki et al. 2007). Further, human endothelial cells produce CYSLTR2 and not CYSLTR1 and IFN-γ enhances CYSLTR2 mRNA production and consequently the CYSLTR2 response to agonist (Woszczek et al. 2007).

2-oxoglutarate receptor 1 (OXGR1, GPR99)

Introduction

Human 2-oxoglutarate receptor 1 (hOXGR1), originally designated as an alpha-ketoglutarate receptor, is a member of the seven transmembrane receptor G-protein coupled receptor 1 family and P2Y subfamily. It binds to and is activated by LTE₄ in the low nM region, AMP and adenosine in the hundreds of nM region, and α-ketoglutarate in the tens of µM range (Fig. 1, Table 4). Stimulation of the OXGR1 receptor leads to an increase in [Ca²⁺]_i and cAMP, indicating that it couples through at least 2 different G-proteins (Inbe et al. 2004). OXGR1 has a role in vascular permeability (Bankova et al. 2016; Maekawa et al. 2008; Kanaoka et al. 2013) bronchoconstriction (Laitinen et al. 1993) and inflammation (Salimi et al. 2017; Steinke et al. 2014).

hOXGR1 (GPR99, CYSLT3, P2Y15, 2-oxoglutarate receptor 1, alpha-ketoglutarate receptor 1, UniProtKB- Q96P68) is translated as a 337 amino acid polypeptide with a calculated molecular weight of 38.3 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 34 residues and a cytosolic C-terminal with 32 residues. There are no additional isoforms and no reported coding SNP variants and one L45 frameshift mutant all with unknown effect on function (Landrum et al. 2016). No x-ray structures are available. There is one model available on the SWISS-Model site using P2Y purinoceptor (35.8% sequence homology; PDB entry 4XNV) as a template (Waterhouse et al. 2018) for a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hOXGR1 is expressed in high amounts in the brain, gastrointestinal system, kidney and bladder, male and female tissues, skin and blood as well as lower expression in other tissues (Uhlén et al. 2015). GPR 99 is a high affinity receptor for LTE₄ with an of EC₅₀ ≤ 10 nM with much lower affinity for AMP, adenosine, and α-ketoglutarate (Table 4). Although CYSLTR1 and CYSLTR2 also bind LTE₄, they do so with much lower efficiency (Fig. 1, Table 4) suggesting that OXGR1 is the more likely LTE₄ receptor. Further support for this comes from studies utilizing CYSLTR1/CYSLTR2 double knockout mice where treatment LTE₄ elicits an elevated vascular leak response (Maekawa et al. 2008). Definitive proof is evident from a study using OXGR1/CYSLTR1/CYSLTR2 triple knockout mice where the LTE₄ response is curtailed by 90% compared to wild-type mice (Kanaoka et al. 2013).

Mechanism of cell activation

To date, few publications present details of specific mechanisms that follow stimulation of OXGR1 and only one describing LTE₄ stimulation. Here, the data indicate that both [Ca²⁺]_i and cAMP increase with LTE₄ stimulation, suggesting that signaling occurs through both Gα_q and Gα_s proteins. The authors note that cAMP activation might be a function of the cell type used for the transfection (Inbe et al. 2004). Other studies show that activation with AMP, adenosine, or α-ketoglutarate also results in increased [Ca²⁺]_i, further supporting a Gα_q signaling pathway (Steinke et al. 2014; He et al. 2004).

There are several publications that outline the response of OXGR1 to ligands other than LTE₄ (see review Rajkumar and Pluznick 2017). However, such findings are beyond the scope of this review and will not be discussed further here.

Regulation

Receptor interaction is involved in the regulation of OXGR1. In CYSLTR1/CYSLTR2 the double knockouts noted above, the effect of LTE₄ is enhanced 64-fold, presumably through the action of OXGR1 (Maekawa et al. 2008). This enhancement strongly suggests that both CYSLTR1 and CYSLTR2 cross-regulate the response of OXGR1 (Kanaoka and Boyce 2014).

OXGR1 expression in human lung mast cells is upregulated in response to allergic reactions (Nishi et al. 2016). The particulars of the process are unknown.

Uracil nucleotide/cysteinyl leukotriene receptor (GPR17)

Introduction

Human uracil nucleotide/cysteinyl leukotriene receptor (hGPR17) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family with dual specificity receptor for both uracil nucleotides and cysteinyl leukotrienes, as might be expected for a receptor so closely phylogenetically related to both CYSLTRs and the P2Y purinergic receptors (Abbracchio et al. 2006). There are apparently different binding sites for these two structurally different effector molecules (Abbracchio et al. 2006). The actions of these effector molecules as agonists remains controversial. In terms of biological function, there is significant support for the involvement of GPR17 in oligodendrocyte differentiation and myelination (Marucci et al. 2016; Lu et al. 2018; Franke et al. 2013; Simon et al. 2016).

hGRP17-1(G-protein coupled receptor 17, P2Y-like receptor, R12, UniProtKB-Q13304) is translated as a 367 amino acid polypeptide with a calculated molecular weight of 37.8 kDa. The basic structure consists of seven transmembrane helices with a large N-terminal extracellular domain of 64 residues and a cytosolic C-terminal with 38 residues. There is one additional isoform (hGPR17-2) that is missing the first 28 residues found in the canonical isoform (hGPR17-1). Agonist binding data has been obtained for both isoforms (Table 4). There are two reported coding SNP variants (T27M and T55M), both with unknown effect on function (Stelzer et al. 2016; Landrum et al. 2016). No X-ray structures are available. There is one model available on the SWISS-Model site using the apelin receptor in complex with agonist peptide (31.5% sequence homology; PDB entry 5VBL) as a template (Waterhouse et al. 2018) that provides a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hGPR17 is primarily expressed primarily in the brain with lower amounts in the GI tract, muscle and female tissue and small amounts in most other tissues (Uhlén et al. 2015). Although GPR17 is expressed throughout the brain the highest expression is found in the cerebral cortex, hippocampal formation, amygdala, thalamus, pons, and medulla. Cellular locations include both the plasma membrane and intracellular vesicles.

hGPR17 activation by both leukotrienes and uridine nucleotides have been reported (Table 4). Leukotrienes LTC₄, LTD₄ and LTE₄ all promote [³⁵S]GTPγS binding to the receptor with EC₅₀ values in the sub-nM to low nM range (Ciana et al. 2006; Pugliese et al. 2009; Daniele et al. 2011). The uridine nucleotides UDP, UDP-glucose (UDP-Glc), and UDP-galactose (UDP-Gal) also promote [³⁵S]GTPγS binding albeit in the tens of nM to tens of µM range depending on the cell system employed (Ciana et al. 2006; Pugliese et al. 2009; Daniele et al. 2011; Lecca et al. 2008). Ciana et al (2006) report that UDP, UDP-Glc, UDP-Gal and LTD₄ all inhibit forskolin induced cAMP formation and increase [Ca²⁺]_i in 30% of the transfected cells. Pugliese et al. (2009) also report an increase in [Ca²⁺]_i upon stimulation with UDP-Glc. Daniele et al. (2014) report an inhibition of forskolin induced cAMP formation upon stimulation with UDP-Glc and LTD₄.

Not all laboratories have been able to reproduce these data. Qi et al. (2013) report that hGPR17 transfected CHO cells stimulated with either LTC₄, UDP, UDP-Glc, or UDP-Gal fail to promote inhibition of forskolin-stimulated cAMP accumulation. Similarly, these effectors did not induce inositol phosphate accumulation in COS-7 or HEK293 cells transfected with hGPR17. Similar results have been reported by Simon et al. (2017), where LTD₄ and the uridine nucleotides failed to exhibit [³⁵S]GTPγS binding, Ca²⁺ release, or changes in the dynamic mass redistribution (DMR) in HEK293, 1321N1, or CHO hGPR17 transfectants. They also provide proof that the aforementioned hGPR17 activity does occur in each of these cell systems by showing that the synthetic agonist MDL29,951 produces them.

The inconsistent data for agonist stimulation of GPR17 has led to the proposal of an alternative biological function for GPR17. Maekawa et al. (2009) report that when CYSLTR1 is co-expressed with GPR17 the two receptors colocalize on the cell surface and that specific [³H]LTD₄ binding to microsomal membranes and LTD₄ elicited ERK phosphorylation is fully inhibited. This and GPR17 knockdown studies indicate a ligand-independent negative regulatory role for GPR17. Similar data has been reported by others (Qi et al. 2013).

Mechanism of cell activation

Although the specific, naturally occurring agonist is in doubt, it is clear that the synthetic agonist MDL29,951 does produce cellular changes when binding to GPR17 and provides insight into the signaling pathways. Hennen et al. (2013) present an extensive study of signaling by hGPR17 transfected into CHO, HEK293, and 1321N1 cell lines (Table 4). In all cases, signaling produces an increase in DMR, [Ca²⁺]_i, inositol phosphate accumulation, and [³⁵S]GTPγS binding. Changes in cAMP are bimodal, where low concentrations of agonist produce a decrease in forskolin-stimulated cAMP accumulation and higher concentrations reverse this trend. In both CHO and HEK293 cells the reversal results in an overall increase in cAMP accumulation over that produced by forskolin induction. Hennen et al. (2013) also characterized the signaling pathways involved in the changes in cAMP, inositol phosphate and [Ca²⁺]_i. Treatment of either HEK293 or CHO cells with PTX eliminates the reduction in cAMP accumulation, indicating that the low agonist concentrations are stimulating signaling through a Gα_i pathway. The increase in cAMP at higher agonist concentrations is indicative of Gα_s-mediated signaling which is supported by the results from Bioluminescence Resonance Energy Transfer (BRET2) assays. They also show that the inositol phosphate accumulation and the increase in [Ca²⁺]_i is Gα_q-mediated, as these increases are blunted by the Gα_q-selective inhibitor FR900359. Simon et al. (2015) found similar results for endogenous GPR17 in murine oligodentrocytes (Oli-neu cells) and confirmed both Gα_i and Gα_q signaling. They did not observe the production of cAMP at higher concentrations of agonist as shown in the aforementioned study. Further, they show that the Gα_i path is responsible for diminished myelin protein accumulation and does so by reducing the activity of a cAMP/PKA/cAMP response element binding protein (CREB) cascade.

Regulation

Receptor regulation by β-arrestin-mediated internalization is a common feature of GPCR regulation. Hennen et al. (2013) show that MDL29,951 stimulates GPR17 and promotes β-arrestin2 recruitment, and that this occurs through two mechanisms, one being G-protein dependent and the other G-protein independent. Further, agonist dependent desensitization by β-arrestin occurs both through the binding of β-arrestin itself and through promotion of internalization following binding. Fratangeli et al. (2013) report that both LTD₄ and UDP-Glc stimulate a clatherin-mediated internalization of GPR17 in Oli-neu cells, some of which is targeted for lysosomal degradation. Daniele et al. (2014) report that both GRK2 and GRK 5 are involved in GPR17 internalization and show that both kinases are weakly associated with GPR17 under basal conditions. Upon LTD₄ binding, GRK2 strongly associates with the receptor resulting in receptor phosphorylation. UDP-Glc binding promotes strong association with GRK5 which also results in receptor phosphorylation. Further they show a weak basal association of β-arrestin with GPR17 and that agonist binding strengthens this association and that the complexes formed through LTD₄ activation are more transient than those stimulated by UDP-Glc binding. They suggest that the difference in binding strength leads to the complexes formed through LTD₄-mediated internalization remaining close to the plasma membrane.

P2Y purinoceptor 12 (P2Y12)

Introduction

Human P2Y purinoceptor 12 (hP2Y12) is a member of the G-protein coupled receptor 1 family and the P2Y (purinergic receptor) subfamily of receptors. It I well known for its involvement in platelet activation (Abbracchio et al. 2006; Bye et al. 2016; Parke and Storey, 2021) and inflammation (Neves et al. 2010;

Paruchuri et al. 2009; Sasaki et al. 2019). As a member of the purinergic receptor family, much of the data available for this receptor centers on purine ligands, where the most effective endogenous agonist is ADP followed by ATP (Burnstock, 2018). However, there are increasing numbers of reports that implicate LTE₄ as a highly effective agonist (Nonaka et al. 2005; Sasaki and Yokomizo, 2019). However, the claim remains controversial (Foster et al. 2013).

hP2Y12 (ADPG-R, P2Y(AC), SP1999, UniProtKB-Q9H244) is translated as a 342 amino acid polypeptide with a calculated molecular weight of 39.4 kDa. The basic structure consists of seven transmembrane helices with a large N-terminal extracellular domain of 64 residues and a cytosolic C-terminal with 21 residues. There are no additional isoforms. There are 35 reported coding SNP variants of which 9 are involved in abnormal platelet function or bleeding disorders (R265P, R265W, P258T, R256Q, I240fs, I200M, C175Y, K174E, R122H) (Landrum et al. 2016). Six X-ray structures are available (e.g., PDB entry 4NTJ). Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hP2Y12 is primarily expressed in the brain with lower amounts in the blood, bone marrow and lymphoid tissues, lung (protein), adipose tissue and small amounts in most other tissues (Uhlén et al. 2015). hP2Y12 is expressed throughout the brain in relatively equal amounts with the exception of the cerebellum where the expression is much lower. It is important to note that neural expression of P2Y12 is exclusive to microglia and it functions to enhance neuropathic pain associated with nerve injury (Gu et al. 2016; Kobayashi et al. 2008). Cellular locations have been confirmed for both the plasma membrane and intracellular vesicles (Mundell et al. 2006).

hP2Y12 activation by both adenine nucleotides and LTE₄ have been reported (Table 4). The adenine nucleotide ADP induces an increase in GTP hydrolysis and inositol phosphate accumulation with EC₅₀ values of 30 and 2.4 µM respectively (Bodor et al. 2003). ADP has also been reported to inhibit forskolin-induced cAMP accumulation with an EC₅₀ of 6.8 nM (Kauffenstein et al. 2004). The rather wide range of efficacies requires further investigation. Nonaka et al. (2005) report a computer modeling study which indicates that LTE₄ should be an effective agonist for P2Y12 and support this assertion with an experimental measurement of LTE₄-induced Ca²⁺ mobilization for which an EC₅₀ of 1.3 nM is apparent. In direct contrast, Foster et al. (2013) report that although they found that ADP and the synthetic agonist 2MeSADP are agonists for hP2Y12, LTE₄ is not an agonist. However, there is considerable support for LTE₄ acting as an agonist for P2Y12. Neves et al. (2010) have reported that both of the leukotriene receptors CYSLTR1 and CYSLTR2 and the hP2Y12 receptor are expressed on eosinophil granule membranes and that LTC₄, LTD₄ and LTE₄ all stimulate isolated granules to secrete eosinophil cationic protein (ECP). They also show that pre-incubation of the granules with the specific P2Y12 antagonist MRS2395 inhibits the release of ECP by each of the three leukotrienes, indicating that hP2Y12 is clearly involved in the process. Pre-incubation with montelukast, a known antagonist for CYSLTR1 exhibits the same effect. These results and the fact that LTE₄ is a poor agonist for CYSLTR1 suggest that the release of ECP in this system may in fact be mediated by a heteromer of hP2Y12 and CYSLTR1. Further support for LTE₄ agonism of P2Y12 is found in the report by Austen et al. (2009). Here they show that application of LTE₄ to sensitized mouse (BALBc) airways induces eosinophilia, goblet cell metaplasia, and the expression of IL-13, a mediator of allergic inflammation and that these effects are completely blocked by the P2Y12-specific antagonist clopidrogel.

Mechanism of cell activation

In contrast to the classic P2Y receptors that couple with Gα_q, P2Y12 couples through the PTX-sensitive Gα_i (Paruchuri et al. 2009; Foster et al. 2001; Bodor et al. 2003). Bodor et al. (2003) examined this coupling in detail and found that the most robust coupling occurs through Gα_i2 but coupling through Gα_i1 and Gα_i3 are also effective. Little if any coupling was observed to occur through Gα_o or Gα_q. An alternative pathway for P2Y12 signaling has also been proposed. Soulet et al. (2004) show that hP2Y12 transfected into CHO cells activates both a cell proliferation pathway and an actin cytoskeleton reorganization pathway. The former pathway proceeds through a Gα_i coupled PI3K/Akt/MAPK pathway whereas the latter is Gα_i-independent and requires activation of RhoA and Rho-kinase.

Regulation

As typical for GPCRs, agonist-induced desensitization and internalization is observed, however, not all reports agree on the degree of desensitization. Hardy et al. (2005) report that the ADP-induced P2Y12 reduction of forskolin-induced cAMP accumulation is blocked by pretreatment with ADP. They further show that this desensitization is mediated by GRKs rather than classical protein kinase C desensitization (PKC). They note that since both GRK2 and GRK6 are endogenously expressed in platelets, these G-proteins are the likely candidates for desensitization. Later work by the same group (Mundell et al. 2006) reveals that desensitization is accompanied by rapid internalization. They show further that the novel PKCγ is capable of facilitating internalization of P2Y12, but not other, classical PKCs. The fact that PKCγ is highly expressed in the human brain, but only marginally expressed blood suggests that this mechanism may play a role in CNS microglia, but not in platelets. In contrast, Baurand et al. (2005) show that preincubation with ADP does not desensitize the P2Y12 reduction in cAMP accumulation. They do observe rapid, transient internalization of P2Y12, but the receptors are rapidly returned to the plasma membrane such that the overall cell surface expression of P2Y12 is unchanged by ADP. Interestingly, the degree of internalization in both studies is remarkably similar (25–30%), suggesting that the rate of surface return may define the difference between the studies. More recently, Nisar et al. (2012) show that the motif-binding protein NHERF1 has a major role in the internalization of P2Y12 and that β-arrestin promotes the interaction between P2Y12 and NHERF1. The relationship between these findings and the earlier reports remains to be seen. Reports of LTE₄-induced internalization have not been published to date.

Lipoxin receptors

Introduction

Lipoxins represent a family of metabolites that are either directly or indirectly produced by lipoxygenases. Lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄) are produced from arachidonic acid first through the action of 5-lipoxygenase to produce LTA₄ that is then converted to both LXA₄ and LXB₄ by 12-lipoxygenase (Fig. 1). Both lipoxins induce anti-inflammatory and pro-resolution mechanisms including repression of leukocyte-mediated injury and pro-inflammatory cytokine production, as well as inhibition of cell proliferation and migration (Tang et al. 1996; Maderna and Godson 2003). These lipoxins are potent activators of monocytes and stimulate chemotaxis and adherence (Maddox and Serhan 1996; Maddox et al. 1997). The 15(R)-epimers of LXA₄ and LXB₄ (15-epi-LXA₄ and 15-epi-LXB₄) are produced from arachidonic acid by aspirin-acetylated cyclooxygenase 2 (COX-2) forming 15(R)-HPETE which then is converted by 5-lipoxygenase into 5(S),6(S)-epoxy-15(R)-ETE and then in turn is enzymatically converted to 15-epi-LXA₄ and 15-epi-LXB₄ (Romano 2010, 2006) (Fig. 1). Although 15-epi-LXA₄ has been shown to possess anti-inflammatory activity like LXA₄ (Romano 2010; Gewirtz et al. 1998; Kain et al. 2017), the anti-inflammatory activity of 15-epi-LXB₄ is somewhat different from that observed for LXB₄ (Maddox et al. 1998). ALX/FPR2 is the primary receptor of LXA₄, however, receptors for LXB₄ or its 15-epi derivative have yet to be identified.

N-formyl Peptide receptor 2 (FPR2/ALX)

Introduction

Human N-formyl peptide receptor 2 (hFPR2/ALX), is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds to and is activated by LTA₄, the aspirin-triggered 15(R)-epimer 15-epi-LTA₄, Resolvin D1 (discussed below), and various signal peptides such as annexin in the low µM region (Figs. 1 and 2). This review will only cover the former oxidized lipids. Readers interested in binding and activation by peptides and proteins are referred to the excellent review by Cattaneo et al. (2013). Stimulation of this receptor with eicosanoids is complex and is dependent on cell type and whether homo- or hetero-dimers are present on the plasma membrane (Filep 2013). The unifying theme, however, is the activation of anti-inflammatory/pro-resolving pathways, including leukocyte trafficking, control of neutrophil apoptosis, and macrophage efferocytosis (Cooray et al. 2013).

hFPR2/ALX (ALX, ALX/FPR2, FPRH1, FPRL1, LXA4R, Lipoxin A₄ receptor, UniProtKB- P25090) is translated as a 351 amino acid polypeptide with a calculated molecular weight of 39.0 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 27 residues and a cytosolic C-terminal with 45 residues. There are no additional isoforms and one coding SNP variant with unknown biological impact reported (Landrum et al. 2016). There are two reported X-ray structures (PDB entry 6LW5 and 6OMM). Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally. However, N-glycosylation in general has been demonstrated for this receptor, but the specific site has not been confirmed (Chiang et al. 2000).

Expression and characterization

hFPR2/ALX is expressed in high amounts in the immune system and in granulocytes as well as lower expression in other tissues including lung and brain (Uhlén et al. 2015). It binds to and is activated by LTA₄, 15-epi-LTA₄ and Resolvin D2 (discussed below) in the sub nM range (Figs. 1 and 2, Table 4). Lipid and peptide ligands bind to distinctively different binding sites (Chiang et al. 2006) for which the lipid binding site has been shown to modulate the signaling initiated by the peptide binding site.

Mechanism of cell activation

The mechanism by which cells respond to hFPR2/ALX stimulation is a function of cell type and is context-dependent in terms of formation of homo- and hetero-dimers on the plasma membrane. For example, when LXA₄ binds to hFPR2/ALX in human monocytes the cells are activated, whereas in neutrophils the cells are inhibited (Maddox et al. 1997), albeit leading to the same result, resolution of inflammation. Only G-protein coupling through Gα_i has been reported for both plasma membrane associated (Le et al. 2002) and nuclear membrane associated hFPR2/ALX where signaling proceeds through an increase in [Ca²⁺]_i. Although all reported signaling mechanisms involve an increase in [Ca²⁺]_i, the calcium source and magnitude of signaling is cell dependent. In human monocytes and goblet cells, a significant portion of the mobilized calcium comes from external sources (Romano et al. 1996; Hodges et al. 2017), whereas in neutrophils [Ca²⁺]_i appear to be regulated intracellularly (Hodges et al. 2017). Complementary to this is the observation, LXA₄ stimulates a robust increase in [Ca²⁺]_i in human monocytes and goblet cells, whereas in PMNs only a small increase in [Ca²⁺]_i is observed (Hodges et al. 2017). Similar results are obtained from rat goblet cells when stimulated with Resolvin D1 (RvD1) where the cellular response is mediated by multiple signaling systems including PLC, phospholipase D, and phospholipase A2 and their signaling components ERK1/2 and calmodulin kinase (Lippestad et al. 2017).

15-epi-LXA₄ has been shown to regulate the response initiated by the synthetic peptide WKYMVm that binds to a separate peptide binding site on hFPR2/ALX (Ge et al. 2020). Utilizing hFPR2/ALX expressed in HEK293 cells, both 15-epi-LXA₄ and WKYMVm were both individually found to reduce cAMP with effective concentrations in the sub nM range, the effect of the former much weaker than the latter. Both were also individually found to increase [Ca²⁺]_i with WKYMVm effective in the sub nM range and 15-epi-LXA₄ only effective in the 10’s of mM range. However, when the cells are challenged with 10 nm WKYMVm, the minimum concentration necessary to elicit the greatest effect on [Ca²⁺]_i and cAMP, increasing concentrations of 15-epi-LXA₄ modulate the WKYMVm signal in a biphasic manner. In the concentration range of 1 to 100 pM 15-epi-LXA₄ reduces the WKYMVm signal to a maximum of 20%. With increasing concentrations in the 1 nm to 1 μM range the WKYMVm signal increased to a maximum of 95% at 1 μM. Clearly 15-epi-LXA₄ serves to modulate the WKYMVm signal in an anti-inflammatory mode where the second phase reflects a weak agonistic activity of the ligand itself.

Agonist binding has been shown to facilitate dimer formation which can lead to different signaling pathways depending on whether the dimer is a homodimer of FPR2/ALX or a heterodimer with some other receptor. Agonist binding to cells transfected with only hFPR2/ALX results in the formation of homodimers which in turn signal through a p38/MAPK/MAPKAPK/Hsp27 signaling pathway, ultimately leading to an increase in the production of IL-10, a known anti-inflammatory cytokine (Cooray et al. 2013; Filep 2013). On the other hand, cells transfected with both hFPR2/ALX and human formyl peptide receptor 1 (FPR1) form heterodimers in the presence of agonist and show a preference for the JNK/Caspase 3 pathway which leads to cell apoptosis (Cooray et al. 2013).

Regulation

N-glycosylation is often found to direct plasma membrane expression and intracellular trafficking of receptors. However, for FPR2/ALX expressed in HEK293 cells, deglycosylation significantly reduces the affinity for peptide substrates, but has no effect on lipid effectors (Chiang et al. 2000). This also supports the observation that peptides and lipids have separate binding sites on this receptor.

Agonist-induced internalization is a common pathway for regulating expression of GPCRs. The two common modes for internalization involve either clatherin-coated pits or the formation of caveolae. LXA₄-induced internalization of murine FPR2/ALX transfected into HEK and human PMN cells proceeds via the formation of caveolae and internalization requires PKC, suggesting a potential receptor phosphorylation event (Maderna et al. 2010). In contrast, FPR2/ALX transfected into HEK293 cells are internalized via clatherin-mediated dynamin-dependent modality when stimulated with a peptide agonist (Huet et al. 2007).

Regulation at the gene level has also been presented. Primary human monocytes produce large amounts of hFPR2/ALX mRNA and express hFPR2/ALX on their plasma membranes whereas macrophages do not. This is due to the fact that monocytes lose the ability to produce hFPR2/ALX upon differentiation into macrophages. However, stimulation of macrophages with either the INF-γ cytokine or LPS results in a > 80% increase in hFPR2/ALX mRNA, but no increase in translation. This loss in ability to produce hFPR2/ALX is explained by the fact that monocytes and macrophages utilize different promotors for hFPR2/ALX transcription, and the promotor used by macrophages results in a longer 5’-UTR that results in reduced translation (Waechter et al. 2012).

Resolvin receptors

Introduction

Resolvins are oxidized products of ω3 PUFAs that are actively involved in the resolution of inflammation. The D series resolvins are derived from eicosapentaenoic acid (EPA), the E series from docosahexaenoic acid (DHA), and the T series from DHA in aspirin-treated cells; aspirin-acetylated COX-2 alters the pathway for the series. A complete description of all these specialized pro-resolving mediators (SPMs) is beyond the scope of this review. Instead, the focus will be on a few classes, namely the Resolvins D1, D2, E1 and E2 which have been studied to the greatest extent.

N-Arachidondyl Glycine receptor, GPR18

Introduction

Human N-arachidondyl glycine receptor (hGPR18), is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds with high affinity and is activated by Resolvin D2 (RvD2), N-arachidondyl glycine (NAGly) and other endocannabinoids, and to a lesser extent, but at biologically relevant concentrations of Δ⁹-tetrahydrocannabinol (Δ⁹-THC). Activation of GPR18 initiates anti-inflammatory/pro-resolving pathways (Serhan and Petasis 2011). The potential for the GPR18 receptor’s role in the pharmacology of pain has also been suggested (Guerrero-Alba et al. 2019).

hGPR18 (GPR18/DRV2, GPCRW, NAGly, UniProtKB- Q14330) is translated as a 331 amino acid polypeptide with a calculated molecular weight of 38.1 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 26 residues and a cytosolic C-terminal with 42 residues. There are no additional isoforms and no coding SNP variants reported (Landrum et al. 2016). There are no reported X-ray structures. There is one potential structural model template, an apelin receptor in complex with agonist peptide (23.9% sequence homology, PDB entry 5VBL) selected by the SWISS-MODEL website (Waterhouse et al. 2018) that should serve as a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hGPR18 is expressed in most tissues with high amounts expressed in lymphoid and bone tissue, and blood with lesser, but significant amounts in testes and vagina (Uhlén et al. 2015). The primary natural agonist for this receptor is RvD2 with a K_d of 9.6 nm, but also binds to Δ⁹-THC with EC₅₀s in the µM range (Fig. 2, Table 5) (Chiang et al. 2015; Rempel et al. 2014; McHugh et al. 2012).

Table 5.

Resolvin receptors

GPR18		Ki /Kd(nM)					References
Receptor/Expression	Parameter measured	RvD2	CBD	Δ9-THC
hGPR18/CHOa	Bind	9.6	–	–	–		Chiang et al. (2015)

EC50/IC50 (nM)
Receptor	Parameter measured	RvD2	CBD	Δ9-THC	NAGly		References
hGPR18/CHOb	β-arrestin	0.0002	–	–	–		Chiang et al. (2015)
hGPR18/CHOb	β-arrestin	–	–	4,610	–		Rempel et al. (2014)
hGPR18/HEK293c	MAPK	–	–	960	–		Rempel et al. (2014)
hGPR18/CHOb	β-arrestin	–	–	14,200	–		Rempel et al. (2013)
hGPR18/HEK293c	MAPK	–	51,000	960	45		McHugh et al. (2012)
hGPR18/CHOd	cAMP↓	–			20		Kohno et al. (2006)

GPR32		Ki /Kd(nM)					References
Receptor/Expression	Parameter measured	RvD1	AT-RvD1	DHA	LXA4
hGPR32/CHOe	Bind	0.17	–	–	–		Krishnamoorthy et al. (2010)
EC50/IC50 (nM)
hGPR32/CHOb	β-arrestin	0.0036	0.0088	0.22	–		Krishnamoorthy et al. (2012)
hGPR32/CHOb	β-arrestin	–	–	–	0.034		Krishnamoorthy et al. (2010)

CMKLR1		Ki /Kd(nM)					References
Receptor/Expression	Parameter measured	RvE1	18(S)-RvE1	RvE2	chemerin	LTB4
hCMKLR1/CHOf	Bind	11.3	–	–	–	–	Arita et al. (2005)
hCMKLR1/CHOf	Bind	330	–	–	429	> 10,000	Arita et al. (2007)
hCMKLR1/CHO-K1g	Disp	–	–	–	0.56	–	de Poorter et al. (2013)
EC50/IC50 (nM)
hCMKLR1/CHOb	β-arrestin	0.137	0.00633	–	–	–	Oh et al. (2011)
hCMKLR1/HEK293h	GTPγS	3	–	–	–	–	Arita et al. (2005)
hCMKLR1/HEK293i	luciferase	6	–	–	20	–	Arita et al. (2005)
hCMKLR1/CHOj	Akt-P	0.5	–	–	0.9	–	Ohira et al. (2010)
hCMKLR1/CHOb	β-arrestin	0.013	–	0.09	–	–	Oh et al. (2012)
hCMKLR1/CHO-K1k	Ca2+↑	–	–	–	0.31	–	de Poorter et al. (2013)

Agonist parameters given for Human (h) receptors in the indicated cell line. Parameter measured: Akt-P, phosphorylation of Akt; β-arrestin, β-arrestin binding; bind, binding assay; Ca²⁺↑, increase in [Ca²⁺]_i; cAMP↓, decrease in forskolin induced cAMP accumulation; disp, displacement assay; GTPγS, binding [³⁵S]GTPγS; luciferase, luciferase assay; MAPK, MAP kinase activation assay. Cell type abbreviations: CHO, Chinese hamster ovary cells; CHO-K1, subclone from the parental CHO cell line; HEK293, Human embryonic kidney 293 cells. Agonist abbreviations: RvE1, resolvin E1; RvE2, resolvin E2; LTB₄, leukotriene B₄. a) K_d for binding [³H]RvD2. b) β-arrestin recruitment assay. c) MAP kinase assay. d) Reduction of forskolin-induced cAMP. e) K_d for binding [³H]RvD1. f) K_d for binding [³H]RvE1. g) Competitive binding of chemerin for bound [¹²⁵I]-chemerin.h) Estimated from plot of [³⁵S]-GTPgS binding. i) Estimated from plot of the inhibition of TNF-α induced NB-κB luciferase activity. j) Estimated from plot for increase in Akt phosphorylation. k) Determined from increase in [Ca²⁺]_i

Mechanism of cell activation

The mechanism by which cells respond to GPR18 stimulation is a function of cell type, cell line, and specific agonist, and is context-dependent with respect to available G-proteins and potential heterodimer partner. When hGPR18 is transfected into CHO cells, stimulation with NAGly leads to an increase in [Ca²⁺]_i and a decrease in cAMP production, indicating coupling through Gα_i due to its PTX sensitivity (Kohno et al. 2006). Although these results have been reported in several publications, there are others that report the inability to initiate signaling by NAGly (Rempel et al. 2014; Finlay et al. 2016). It has been suggested that the lack of activity might be due to the absence of a required interacting protein in the system utilized (Finlay et al. 2016). Stimulation with RvD2 appears to be coupled through a Gα_s-like proteins due to its cholera toxin (CTX) sensitivity (Chiang et al. 2015). In mouse and human macrophages, RvD2 stimulation of GPR18 results in an increase in cAMP, fostering a cAMP-PKA signaling pathway that is also coupled through a Gα_s, leading to enhanced macrophage phagocytosis (Chiang et al. 2017). The same RvD2-induced signaling pathway is also observed in rat conjunctival goblet cells (Botten et al. 2019).

As observed for other eicosanoid receptors, GPR18 can form heterodimers with other receptors. For example, hGPR18 forms a heterodimer with the human cannabinoid receptor CB2R when co-transfected. Rather than exhibiting a synergic effect upon stimulation with cannabinoids as observed for CB2R transfected along with CB1R, the GPR18/CB2R heterodimer exhibits a reduced response (Reyes-Resina et al. 2018).

As noted above, RvD2/GPR18 signaling proceeds through a cAMP/PKA pathway. This in turn leads to enhanced phosphorylation of cAMP response element-binding protein (CREB), ERK1/2 and the signal transducer and activator of transcription 3 (STAT3). PKA and STAT3 notably enhance macrophage phagocytosis of bacteria (Chiang et al. 2017) and PMN apoptosis as well as reducing PMN infiltration (Chiang et al. 2015) in keeping with this receptor’s involvement in resolution of inflammation. Further, in a murine cecal ligation and puncture (CLP) model, RvD2 stimulation reduces levels of inflammatory cytokines IL-6, IL-10, IL-1β, IL-23, IL17 and TNF α as well as reducing pro-inflammatory mediators PGE₂ and LTB₄ (Spite et al. 2009). NAGly stimulation of GPR18 also enhances production of LXA₄ (Chiang et al. 2015). A known pro-resolving, anti-inflammatory eicosanoid.

Regulation

Regulation of GPR18 is not clearly understood. β-arrestin binding, a common mode to initiate agonist-induced receptor internalization, is a function of which agonist binds to GPR18. Both RvD2 (Chiang et al. 2015) and Δ⁹-THC initiate β-arrestin binding whereas NAGly does not (Console-Bram et al. 2014; Yin et al. 2009). To date, neither quantitation of nor mechanism for the event has been reported. However, hGPR18 expressed in HEK cells exhibits a rapid, constitutive trafficking from cytosolic stores to the plasma membrane, however, the surface expression is significantly lower than the total hGPR18 cellular content suggesting some form of control mechanism (Finlay et al. 2016).

Transcriptional regulation of GPR18-like signaling is also cell type dependent. mGPR18 mRNA expression in mouse macrophages is inducible and increases 52-fold after treatment of murine bone marrow-derived macrophages (BMM) with LPS, whereas the expression in murine thioglycolate-elicited peritoneal macrophages (TEPM) is unaffected and thus constitutively expressed (Lattin et al. 2008). Out of 75 orphan GPCRs, hGPR18 is the most abundantly over-expressed GPCR in human melanomas. Further, hGPR18 is constitutively active in melanomas and serves to inhibit apoptosis, indicating that it has an important role in the tumor cell survival (Qin et al. 2011).

G-Protein receptor 32, GPR32

Introduction

Human G-protein Receptor 32 (hGPR32) is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds with high affinity and is activated by Resolvin D1₁ (17S, RvD1) and its aspirin-triggered 17R isomer AT-RvD1, both in the pM range, as well as LXA₄ and DHA in the sub nM range (Table 5, Figs. 1 and 2). Activation initiates anti-inflammatory/pro-resolving pathways. (Krishnamoorthy et al. 2010; Krishnamoorthy et al. 2011; Serhan et al. 2011).

hGPR32 (Probable G-protein coupled receptor 32, DRV1, UniProtKB- O75388, UniProtKB- H9NIL6) is translated as a 356 amino acid polypeptide with a calculated molecular weight of 40.1 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 44 residues and a cytosolic C-terminal with 36 residues. There are no additional isoforms and one coding SNP variant F327L with unknow biological effect (Stelzer et al. 2016). No X-ray structures are available. There is a potential structural model template cryo-EM structure of formyl peptide receptor 2/lipoxin A₄ receptor in complex with Gα_i (40.7% sequence homology, PDB entry 6OMM) selected by the SWISS-MODEL website (Waterhouse et al. 2018) that should serve as a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally.

Expression and characterization

hGPR32 is expressed high amounts in parathyroid tissue and low amounts in male and female tissue and cells of the immune system (http://proteinatlas.org, Uhlén et al. 2015). The primary natural agonists for this receptor are RvD1 with an EC₅₀ of 3.6 pM, AT-RvD1 with an EC₅₀ of 8.8 pM, LXA₄ with an EC₅₀ of 34 pM, but also binds DHA with an EC₅₀ of 220 pM (Figs. 1 and 2, Table 5) (Krishnamoorthy et al. 2010, 2012).

Mechanism of cell activation

Activation of hGPR32 with RvD1 in human PMNs blocks actin polymerization (Krishnamoorthy et al. 2010) thereby reducing mobility as expected for a pro-resolving response. Sensitivity to PTX and insensitivity to CTX indicates that the signaling likely proceeds through a Gα_i/o mediated pathway. No change in [Ca²⁺]_i is observed as expected for the proposed path, but no change in cAMP levels is observed as would be expected for this pathway. However, RvD1 stimulation of human macrophages that naturally express hGPR32 results in an increase in [Ca²⁺]_i and a change in phenotype towards a pro-resolving M2 phenotype with a reduction in secreted pro-inflammatory cytokines (e.g., IL-1β and IL-8), enhanced phagocytosis, and blocked chemotaxis towards chemoattractants (Schmid et al. 2016). RvD1 stimulation of hGPR32 transfected into human macrophages also enhances phagocytosis (Krishnamoorthy et al. 2010). Similarly, RvD1 stimulation of human goblet cell GPR32 activates extracellular EGFR and phospholipases D, C and A2 which lead to an increase in [Ca²⁺]_i (Dartt et al. 2019). Stimulation also results in activation of extracellular regulated kinase (ERK) 1/2 leading to mucin secretion into the tear film to protect ocular tissue from the environment. At the same time, both RvD1 and AT-RvD1 stimulate GPR32 in conjunctival goblet cells to prevent histamine-stimulated H1 receptor response thus reducing inflammation (Li et al. 2013). Clearly, signaling through GPR32 is complex and at the very least, dependent on cell type and perhaps yet-to-be discovered partners in heteromers.

As noted above, the expression of GPR32 in parathyroid tissue far exceeds that of any other tissue. However, to date there are no reports describing the mechanism or action of GPR32 signaling in this tissue. The only relationship thus far elucidated is that parathyroid hormone (PTH), the major product of the parathyroid gland induces the production of resolvins, including RvD1 to induce efferocytosis of apoptotic osteoblasts (McCauley et al. 2014). It is possible that PTH localized about the parathyroid glands may induce the same effect to prevent the onset of inflammation-induced hyperthyroidism. See Talat et al. (2011) for a discussion of inflammation of the parathyroid gland.

Regulation

Regulation of GPR32 has yet to be described in detail. It is known that RvD1 stimulates receptor reuptake of both hGPR32 and hFPR2/ALX, albeit the uptake of hGPR32 occurs at lower concentrations of agonist and hFPR2/ALX uptake occurs only after stimulation with TNF-α. Interestingly, the surface expression of hFPR2/ALX, but not hGPR32 is up-regulated in the presence of pro-inflammatory regulators (e.g., TNF-α and IL-8). This suggests that hGPR32 is constitutively expressed to convey normal physiological functions (Norling et al. 2012) and hFPR2/ALX responds to acute conditions. In direct support of this hypothesis, the pro-inflammatory mi-RNA-181b down-regulates the surface expression of hFPR2/ALX on human macrophages but has no effect on hGPR32 surface expression (Pierdomenico et al. 2015).

Chemokine-like receptor 1 (CMKLR1)

Introduction

Human Chemokine-like receptor 1 (hCMKLR1), is a member of the seven transmembrane receptor G-protein coupled receptor 1 family. It binds with high affinity and is activated by Resolvin E₁ (18(S)-Resolvin E₁, RvE1), its aspirin-triggered 18(R)-isomer AT-RvE1 with EC₅₀s in the sub-nM and pM range respectively as well as the chemerin peptide in the low nM range (Table 5, Fig. 2) (Oh et al. 2011; Arita et al. 2005; Ohira et al. 2010). Noteworthy is the fact that although RvE1 binds to the LTB₄ receptor LTB4R with a comparable EC₅₀ and that LTB₄ binds poorly to hCMKLR1 (Serhan et al. 2011).

hCMKLR1 (hChemR23, DEZ, UniProtKB- Q99788) is translated as a 373 amino acid polypeptide with a calculated molecular weight of 42.3 kDa. The basic structure consists of seven transmembrane helices with a N-terminal extracellular domain of 41 residues and a long cytosolic C-terminal with 53 residues. There is one confirmed additional isoform (isoform B) that is missing residues 1–2 and there are two coding SNP variants with unknown biological impact reported: M287L, and M289I (Landrum et al. 2016). There are no reported X-ray structures. There is a potential structural model template cryo-EM structure of formyl peptide receptor 2/lipoxin A4 receptor in complex with Gα_i (27.8% sequence homology, PDB entry 6OMM) selected by the SWISS-MODEL website (Waterhouse et al. 2018) that should serve as a working model. Predicted posttranslational modifications (PTMs) are presented in the supplement Table S1, none of which have been confirmed experimentally. Only indirect evidence for phosphorylation at S345 and S349 has been presented (Oh et al. 2012).

Expression and characterization

hCMKLR1 is expressed in most tissues with particularly high expression in bone and lymphoid tissue, brain, blood and female tissues (Uhlén et al. 2015). The primary lipid-derived natural agonists are RvE1, AT-RvE1, and RvE2 with EC₅₀s in the sub-nM to low nM range (Fig. 2, Table 5). The peptide chemerin is also a strong agonist with EC₅₀s in the nM range.

Mechanism of cell activation

The cellular response to CMKLR1 activation is agonist and cell-type dependent. Activation of CMKLR1 by either RvE1 or RvE2 elicits similar pro-resolving effects, however, RvE2 is less efficacious and less potent than RvE1, suggesting that this receptor might not be the primary receptor for RvE2 (Oh et al. 2012; Tjonahen et al. 2006). The peptide chemerin can be competed out by RvE1 for hCMKLR1 expressed in HEK cells, indicating that the two agonists bind to the same site on the receptor, however, the response from the chemerin-induced G-protein coupling is nearly 3 times larger than RvE1 and induces a different cellular response. Further, RvE1 stimulates strong NF-κB inhibition and does not promote receptor-initiated increases in extracellular acidification rates (EARs) whereas chemerin does evoke EARs and is ten times weaker in NK-κB inhibition (Arita et al. 2005). Additionally, hCMKLR1 is naturally expressed in human chondrocytes where it initiates a pro-inflammatory rather than a pro-resolving response, producing enhanced levels of pro-inflammatory cytokines including IL-6, IL-8, TNF-α and IL-1β (Berg et al. 2010). Murine plasmacytoid dendridic cells (pDCs) express mCMKLR1 and are recruited to areas of infection by chemerin released by cells in the affected area where the pDCs release the interferons INF-α and INF-β that serve to enhance the inflammatory response (Bondue et al. 2011).

The signaling pathways for RvE1 are not well defined. Thus far it has been shown that RvE1 stimulation of hCMKLR1 expressed in CHO cells do signal through a PI3K/Akt/mTOR pathway (Ohira et al. 2009). Further, hCMKLR1 signaling is PTX sensitive suggesting a Gα_i dependent signaling pathway (Arita et al. 2005). Signaling pathways for chemerin, however, are considerably more well defined. Human chemerin and various C-terminal peptides of chemerin (e.g. chemerin 9) stimulate hCMKLR1 with EC₅₀s in the low nM range which lead to coupling with Gα_i/o resulting in an increase in [Ca²⁺]_i (Wittamer et al. 2004). It has since been shown that human chemerin 9 (residues 149–157) produces coupling to three subtypes of Gα_i (Gα_i1, Gα_i2, Gα_i3) and two isoforms of Gα₀ (Gα_0a, Gα_0b) (De Henau et al. 2016). In human fibroblast-like synoviocytes (FLS) taken from rheumatoid arthritis patients express hCMKLR1 and when stimulated with chemerin, activation of ERK1/2, p38MAPK and Akt is enhanced which in turn induces IL-6 production (Kaneko et al. 2011).

Regulation

Increased hCMKLR1 transcription and subsequent protein expression increases during the differentiation of monocytes to macrophages and is further enhanced in classically activated human M1 macrophages. Further, transcription levels increase after treatment of human monocytes with LPS or inflammatory cytokines (Herová, 2015). Similar results have been reported by others (Arita et al. 2005).

Agonist-induced internalization of CMKLR1 by RvE1 or RvE2 is currently unknown. However, Chemerin-induced internalization has been examined. Downregulation of the surface expression for hCMKLR1 by chemerin is facilitated through an agonist-dependent and clatherin-independent process. Chemerin and the active chemerin-derived nonapeptide chemerin 9 induce internalization of hCMKLR1 expressed in HEK293 cells in a dose-dependent manner with chemerin 9 causing up to 90% internalization (Zhou et al. 2014). Ser to Ala mutations of six potential phosphorylation sites reveal that phosphorylation of S345 and S349 (S343 and S347 in isoform B) are required for internalization to occur. Internalization proceeds through a caveolae-mediated rather than a clatherin-mediated process (Oh et al. 2012). Chemerin 9 and chemerin stimulation of hCMKLR1 expressed in CHO-K1 promotes binding of β-arrestin 1 and 2 as well as internalization, suggesting an arrestin-mediated, clatherin-dependent internalization (De Henau et al. 2016). However, the kinetics of arrestin binding and receptor down-regulation do not correlate, the latter being faster, suggesting another mode for internalization is at work.

Other resolvin effects

Resolvins also serve as agonists, antagonists or antagonist/partial-agonists for several receptors. RvD1 is a potent agonist for FPR2/ALX (see above) with EC₅₀ values comparable to that of the primary agonist LXA₄ (Lippestad et al. 2017; Krishnamoorthy et al. 2012, 2010). RvE1 and RvE2 serve as equally potent antagonist/partial-agonists for the LTB₄ receptor LTB4R and thus compete with LTB₄ but activate the receptor to a lesser extent than LTB₄ (Fig. 1, Table 2) (Oh et al. 2012). This suggests that the reduced potency/efficacy of RvE2 may merely reflect a similar antagonist/partial-agonist activity for the CMKLR1 receptor. Interestingly, LTB₄ does not bind to CMKLR1 (Arita et al. 2007). RvD1, RvD2 and RvE1 differentially inhibit capsaicin-induced pain through the TRPV1 and TRPA1 receptors. RvD2 is the most potent antagonist with an EC₅₀ of 0.1 nM for TRPV1 and 2.1 nM for TRPA1 (Park et al. 2011).

Hepoxilin and Trioxillin receptors

Introduction

Hepoxilins are short-lived monohydroxy-epoxides formed from the reactive 12(S)- and 12(R)-HPETE products of ALOX12 and ALPX12B respectively to Hepoxilin B₃ (HxB₃) and Hepoxilin A₃ (HxA₃) respectively (review, Biringer 2018). Hepoxilins are readily converted to their tri-hydroxy counterparts, trioxilins, spontaneously or through the action a soluble epoxy-hydrolases (Cronin et al. 2011). Hepoxilins stimulate both mobilization of intracellular calcium in neutrophils and enhancement of plasma leakage (Pace-Asciak 2015). Hepoxilins also exhibit significant chemotaxis abilities. For example, gradients of HxA₃ have been shown to attract neutrophils across epithelial barriers (Kubala et al. 2014). Trioxilins on the other hand, namely TrXA₃ and TrXC₃ and HxA₃ act as thromboxane antagonists and thus help regulate vascular homeostasis (Siangjong et al. 2017).

The receptors for Hepoxilin and Trioxilin

Specific receptors for hepoxilin agonism have been reported. Both HXA₃ and HXB₃ trigger sustained calcium mobilization in both CHO cells overexpressing each TRPV1 (discussed above) and TRPA1 receptors and in rodent sensory neurons. The net result of hepoxilin agonism is a profound, persistent tactile allodynia as well as a small, transient heat hyperalgesia (Gregus et al. 2012).

Both hepoxilins and trioxilins bind to thromboxane receptors (see Biringer 2020) and serve to function as natural antagonists rather than agonists (Fig. 1, Table 6). Both classes of molecules have been shown to be antagonists of the Thromboxane receptor TP (Fig. 1, Table 6). The stable hepoxilin analog PBT-3 ([10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-treinoic acid methyl ester) has been shown to effectively compete with the TP agonists I-BOP and U46619 on human platelet TP receptors and reduces agonist-induced platelet aggregation (Pace-Asciak et al. 2002). PBT-3 antagonizes I-BOP induced intracellular calcium release in human platelets with an IC₅₀ of 7 nM. Further, PBT-3 selectively antagonizes the TPα isoform. In transfected COS-7 cells it competes with the strong TP antagonist [³H]SQ 29,548 with an IC₅₀ of 200 nM for the TPα isoform and an IC₅₀ of 1200 nM for the TPβ isoform; the [³H]SQ 29,548 has similar K_d values for each receptor (K_d = 11–12 nM) (Qiao et al. 2003). Similarly, trioxilin A₃ (TXA₃) and trioxilin B₃ (TXB₃) antagonize the U46619-induced intracellular calcium increase in HEK293 cells overexpressing human TPα (Siangjong et al. 2017). TXA₃, TXB₃, and trioxilin C₃ (TXC₃) induce vasodilation in U46619-constricted mouse mesenteric arteries with EC₅₀ values of 1.26, 7.15, and 1.00 µM respectively. Cis-epoxy isomers of HXA₃ (11(R),12(S)- and 12(R),11(S)-HXA3) also vasodilate U46619-constricted mouse mesenteric arteries (Siangjong et al. 2017).

Table 6.

Trioxilin receptors: TP

Receptor/Expression	Parameter measured	IC50/EC50(nM)						References
		PBT-3	11(S),12(R)-HxA3	11(R),12(S)-HxA3	TrXA3	TrXB3	TrXC3
hTP/human plateletsa	disp	8.1	–	–	–	–	–	Pace-Asciak et al. (2002)
hTP/human plateletsb	aggregation	63	–	–	–	–	–	Pace-Asciak et al. (2002)
hTP/human plateletsc	Ca2+↓	7	–	–	–	–	–	Qiao et al. (2003)
hTPa/COS-7d	disp	200	–	–	–	–	–	Qiao et al. (2003)
hTPb/COS-7d	disp	1200	–	–	–	–	–	Qiao et al. (2003)
mTP/MMAe	vaso	–	6300	6300	1260	7150	1000	Xue et al. (2011)

Agonist parameters given for Human (h) and murine (m) receptors in the indicated cell line. Parameters measured: aggregation, platelet aggregation assay;disp, displacement assay; Ca²⁺↓, inhibition of Ca2 + release; vaso, vasorelaxation assay. Cell type abbreviations: COS-7, African green monkey kidney cell line; MMA, mouse mesenteric arteries. Agonist abbreviations: HxA₃, hepoxilin A₃; PBT-3: stable hepoxilin analog [10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z trienoic acid methyl ester; TrXA₃, trioxilin A₃; TrXB₃, trioxilin B₃; TrXC₃, trioxilin C₃. a) IC₅₀ for displacement of ¹²⁵I-BOP. b) IC₅₀ for inhibition of I-BOP-induced platelet aggregation. c) IC₅₀ for inhibition of calcium release by I-BOP. d) IC₅₀ for displacement of the TP antagonist [³H]29,548 (K_d = 11–12 nM). e) EC₅₀ for vasorelaxation of U46619 (stable TP agonist) induced vasoconstriction of mouse mesenteric arteries (MMA)

Conclusions

In this review a summary of what is known about the expression, characterization, regulation, and mechanism of action of non-prostanoid, eicosanoid receptors with a focus on human receptors is presented. These receptors control numerous biological functions not only through the diversity in eicosanoids themselves, but through the diversity of receptors, heterodimers, and the heterogeneity of G-protein coupling. Commonality is found in regulation where agonist-induced receptor desensitization is accomplished by phosphorylation by PKA, PBK, PKC or a variety of different GRKs. Although much is known about eicosanoid receptor signaling, there are still many missing pieces, in particular with respect to posttranslational modifications and the associated functions thereof. It is our hope that this review will help emphasize these areas and stimulate research to identify these missing pieces of the puzzle.

Supplementary Information

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Declarations

Conflict of Interest

The author declares that there is no conflict of interest.