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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2014 Sep 5;172(3):785–798. doi: 10.1111/bph.12801

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants

D Wifling 1, K Löffel 1, U Nordemann 1, A Strasser 1, G Bernhardt 1, S Dove 1, R Seifert 2, A Buschauer 1
PMCID: PMC4301689  PMID: 24903527

Abstract

Background and Purpose

Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4R) and rat H4 receptor (rH4R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M; double mutants: hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S.

Experimental Approach

Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([3H]-histamine), and in functional [35S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor.

Key Results

Constitutive activity decreased from the hH4 receptor via the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity.

Conclusions and Implications

F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc

Table of Links

TARGETS LIGANDS
H4 receptor Clobenpropit
H1 receptor Clozapine
H3 receptor Histamine
β2-adrenoceptor Immepip
Thioperamide
JNJ7777120
VUF8430
GTPγS
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This Table lists key protein targets and ligands in this document, which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).

Introduction

The human histamine H4 receptor (hH4R) was independently discovered by several groups (Nakamura et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Oda and Matsumoto, 2001; Zhu et al., 2001). The H4 receptor is coupled to Gαi proteins, leading to inhibition of adenylyl cyclase and, via release of Gβγ complexes, to the activation of phospholipase C (for reviews, see, e.g. Thurmond et al., 2008; Leurs et al., 2009; Seifert et al., 2013). H4 receptor-mediated Gαi activation in membrane preparation is monitored by agonist-stimulated [35S]-GTPγS binding to Gαi proteins or Gαi-mediated [γ-32P]-GTP hydrolysis (Schneider et al., 2009). The H4 receptor is primarily expressed in cells of the immune system and seems to play a pro-inflammatory role in bronchial asthma, atopic dermatitis and pruritus (de Esch et al., 2005; Dunford et al., 2006; Zampeli and Tiligada, 2009; Dunford and Holgate, 2011; Marson, 2011; Schnell et al., 2011). Human H4 receptor expression and function has been unequivocally demonstrated by several independent groups in eosinophils (O'Reilly et al., 2002; Buckland et al., 2003; Ling et al., 2004; Reher et al., 2012). However, eosinophils are very difficult to purify in sufficient amounts for pharmacological studies so that experiments with recombinant hH4 receptor are very important.

A GPCR capable of producing its biological response in the absence of a bound ligand is termed constitutively active (Seifert and Wenzel-Seifert, 2002). Previous studies have shown that the hH4 receptor possesses an unusually high constitutive activity, resulting in high agonist-independent Gαi protein activation (Morse et al., 2001; Seifert et al., 2013; Strasser et al., 2013). A plausible cause could be the missing ionic lock between an arginine in the DRY motif (TM3) and an acidic amino acid in TM6 (replaced by an alanine in the hH4 receptor). However, this was not confirmed by reconstitution of this motif in the hH4 receptor (Schneider et al., 2010). The constitutive activity of canine, murine and rat H4 receptor species isoforms (cH4, mH4 and rH4 receptor, respectively) is substantially lower (Schneider et al., 2010; Schnell et al., 2011; Strasser et al., 2013). Another striking difference was observed with the prototypical H4 receptor antagonist JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine, Figure 1), a partial agonist at the cH4 receptor, the rH4 receptor and the mH4 receptor, but a partial inverse agonist at the hH4 receptor. Furthermore, H4 receptor agonists (Igel et al., 2010) from the class of NG-acylated imidazolylpropylguanidines and cyanoguanidines differed with respect to affinity, potency and efficacy among H4 receptor species isoforms (Schnell et al., 2011).

Figure 1.

Figure 1

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Structures of H4 receptor ligands investigated.

Mouse, rat and dog are important laboratory animal species for assessing the pathophysiological role of the H4 receptor (Liu et al., 2001b; Dunford et al., 2006; Rossbach et al., 2009). It is therefore important to characterize the effects of ligands at those H4 receptor species orthologues in comparison to the hH4 receptor. Considering the rather low sequence identity of H4 receptor species isoforms (see alignment of hH4 receptor, mH4 receptor and rH4 receptor, Figure 2), the question arises which molecular determinants account for the species differences in constitutive activity, ligand binding and intrinsic activity.

Figure 2.

Figure 2

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Sequence alignment of hH4, rH4 and mH4 receptors. TMs are indicated by wavy lines. N-term, N-terminus (extracellular); C-term, C-terminus (intracellular); ICL1, ICL2 and ICL3: first, second and third intracellular loops; ECL1, ECL2 and ECL3: first, second and third extracellular loops. Dots in the sequences indicate identity with the hH4 receptor. The most conserved residues in each TM domain are highlighted by grey shading. TMs were defined according to the hH4 receptor homology model based on the crystal structure of the hH1 receptor.

A systematic investigation with chimeras localized the region between V1414.51 and E1825.46 [superscripts according to the Ballesteros and Weinstein numbering (Ballesteros and Weinstein, 1995)] involving the second extracellular loop (ECL2) to be responsible for differences in agonist affinity between the hH4 receptor and the mH4 receptor (Lim et al., 2008). Moreover, among single hH4 receptor-mH4 receptor amino acid exchanges in this region, the hH4R-F169V mutant resulted in the largest shifts towards the Kd and pKi values at the mH4R, suggesting that this residue in ECL2 is ‘the key amino acid’ for differential interactions of certain agonists with the hH4 receptor and the mH4 receptor (Lim et al., 2008). As in the case of the two corresponding consecutive phenylalanine residues in the β2-adrenoceptor structure (Cherezov et al., 2007), it was assumed that F169 is involved in a network of hydrophobic interactions, stabilizing ECL2 in a conformation, which positions F168 towards the binding pocket (Lim et al., 2008).

To further investigate the role of F169, we generated the single mutants hH4R-F169V and mH4R-V171F. Until now, no functional studies on G-protein coupling at these mutants exist to discriminate between agonist, antagonist and inverse agonist effects. We therefore tested H4 receptor ligands with different qualities of action in functional [35S]-GTPγS assays.

Figure 3 shows the putative histamine binding pocket of the hH4 receptor and various amino acids in the vicinity of this pocket which are specific for the hH4 receptor compared to the mH4 receptor and the rH4 receptor. According to results from in vitro mutagenesis (Shin et al., 2002), the positively charged amino group of histamine forms a salt bridge with D943.32. The ethylamine side chain is embedded between Y953.33 and Y3196.51, whereas the Nπ nitrogen of the imidazolyl moiety is hydrogen bonded to the side chain of E1825.46. F168 (ECL2) points into the binding pocket, albeit direct contacts with histamine are not obvious. Hydrogen bonds of the Nτ nitrogen with the hydroxyls of T1785.42 and S1795.43 are possible, but on the single mutants hH4R-T178A, hH4R-S179A (Shin et al., 2002) and hH4R-S179M (Lim et al., 2008), histamine affinity and activity was only slightly reduced compared to the wild-type hH4 receptor (factors 2–4). However, S1795.43 is mutated in the mH4 receptor (M) and the rH4 receptor (A) and therefore a promising candidate for more detailed investigations. In order to study the pharmacological profile including the constitutive activity of the single hH4R-S179A and hH4R-S179M mutants, we expressed these constructs in Sf9 cells.

Figure 3.

Figure 3

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Ligand binding pocket of the hH4 receptor in complex with histamine. The model is based on the crystal structure of the hH1 receptor as template (Shimamura et al., 2011). Histamine (ball and stick model) was manually docked considering interactions with the hH4 receptor suggested from results of in vitro mutagenesis. Colours of atoms if not otherwise indicated: C – grey, N – blue, O – red, S – yellow. Carbons and backbone nitrogens of amino acids which are different in the rH4 receptor and mH4 receptor are orange-coloured. Other important amino acids of or close to the ligand binding pocket are represented by cyan-coloured C and backbone N atoms. TMs are drawn as ribbons: TM2 – orange, TM3 – yellow, TM5 – green, TM6 – light blue, TM7 – magenta. The C-terminal part of ECL2 is shown as tube.

Although our hH4 receptor model does not indicate direct interactions of S1795.43 and F169 (Figure 3), the question arose whether there is an additive effect of both amino acids with respect to the selectivity of ligands for the human H4 receptor orthologue. We therefore prepared the double mutants of the hH4 receptor, hH4R-F169V+S179A and hH4R-F169V+S179M, corresponding to the rat and mouse H4 receptor in positions 169 and 179, as well as the reciprocal double mutant of the mH4 receptor, mH4R-V171F+M181S.

Methods

Homology model of the hH4 receptor

To suggest promising mutants and hH4 receptor-specific intramolecular interactions close to the ligand binding site, a homology model of the hH4 receptor was generated with the modelling suite Sybyl 7.3 (Tripos Inc., St. Louis, MO, USA) using the crystal structure of the hH1 receptor (protein databank code 3RZE) as template (Shimamura et al., 2011). For this purpose, the inactive state of the template is not inconsistent with the constitutively active state of the hH4 receptor since the binding pocket regions and extracellular domains of both states are very similar (Rasmussen et al., 2011). The resulting model contains all extracellular (ECL) and intracellular (ICL) loops except ICL3 (G215-H292). To close the gap between the intracellular parts of TM5 and TM6, eight alanines were inserted in place of ICL3 (and the lysozyme domain of the template structure respectively). Fifteen missing amino acids of the N-terminus were added by a recently established protocol (Strasser and Wittmann, 2013). The E2 loop is not completely resolved in the hH1 receptor structure. After removing the hH1 receptor residues W165, N166 and H167, the missing amino acids V153-K158 were included into the hH4 receptor model using the Loop-Search module within Sybyl. The inserted regions of the N-terminus and ECL2 were separately refined by energy minimization and a short gas phase MD simulation (500 ps). Histamine was manually docked considering interactions with the hH4 receptor suggested from results of in vitro mutagenesis (Shin et al., 2002). Finally, the model was provided with Amber7 FF99 (histamine: Gasteiger-Hueckel) charges and energy minimized with the Amber7 FF99 force field (Cornell et al., 1995) and a dielectric constant of 4 up to a gradient of 0.01 kcal·mol−1·Å−1.

Miscellaneous

Protein concentrations of all membrane preparations were determined with the Bio-Rad DC protein assay kit (München, Germany) in one experiment. Because UR-PI376 had to be dissolved in 20% DMSO, the water control and the full agonist histamine (α = 1.0), to which all other ligands were referenced, were also dissolved in 20% DMSO in case of this ligand. Data from the [3H]-histamine saturation binding, [3H]-histamine competition binding and the [35S]-GTPγS assays were analysed with the Prism 5.01 software (GraphPad, San Diego, CA, USA). Kb and Ki values were calculated according to the Cheng–Prusoff equation (Cheng and Prusoff, 1973). All values are given as mean ± SEM of at least three (up to nine) independent experiments performed in triplicate. Significances were calculated using one-way anova, followed by Bonferroni's multiple comparison test.

Materials

The pcDNA3.1 vector containing the hH4 receptor sequence was obtained from the UMR cDNA Resource Center at the University of Missouri-Rolla (Rolla, MO, USA). The cDNAs encoding the mouse and rat H4 receptors were a kind gift of Dr R. Thurmond (Johnson & Johnson Pharmaceutical R&D, San Diego, CA, USA). The construction of the human, mouse and rat pVL1392-SF-H4R-His6 and of the pGEM-3Z-SF-mH4R-His6 plasmids, respectively, was described previously (Schneider et al., 2009; Schnell et al., 2011). Baculovirus encoding Gαi2 was kindly provided by Dr A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX, USA). Recombinant baculovirus encoding the unmodified version of the Gβ1γ2 subunits was a kind gift of Dr P. Gierschik (Department of Pharmacology and Toxicology, University of Ulm, Ulm, Germany). Pfu Ultra II DNA polymerase was obtained from Agilent (Böblingen, Germany). The DNA primers for polymerase chain reaction were synthesized by MWG-Biotech (Ebersberg, Germany). Restriction enzymes and T4-DNA ligase were from New England Biolabs (Ipswich, MA, USA). Gradient gels (8–16%, 12 well nUView gels), the ‘prestained’ peqGOLD protein marker III, used for Western blotting as well as the ‘unstained’ peqGOLD protein marker I, used for Coomassie brilliant blue R staining, were from Peqlab (Erlangen, Germany). The antibody selective for Gαi1/2 was from Calbiochem (Darmstadt, Germany). The anti-FLAG M1 antibody, the amino-terminal FLAG-BAP fusion protein and histamine were from Sigma-Aldrich (Taufkirchen, Germany). The binding of secondary antibodies coupled to peroxidase (HRP) was detected with the ECL Western Blotting Substrate (Thermo Scientific, Nidderau, Germany). UR-PI294 and UR-PI376 were synthesized as described previously (Igel et al., 2009a,b,). Thioperamide, JNJ7777120 and VUF8430 were synthesized according to Lange et al. (1995), Jablonowski et al. (2003) and Lim et al. (2006). Isoloxapine (Schmutz et al., 1967; Smits et al., 2006) was provided by S. Gobleder (Institute of Pharmacy, University of Regensburg, Regensburg, Germany). All other H4 receptor ligands were purchased from Tocris (Avonmouth, Bristol, UK). The chemical structures of the ligands are depicted in Figure 1. UR-PI376 (10 mM) was dissolved in 50% (v v−1) DMSO and dilutions were prepared in 20% (v v−1) DMSO in order to attain a final DMSO concentration of 2% (v v−1) in each well. About 10 mM stock solutions of clozapine and isoloxapine were prepared in Millipore water containing 3 and 2 mole equivalents of HCl respectively. All other stock solutions were prepared with Millipore water. [35S]-GTPγS (≥1000 Ci·mmol−1, radiochemical purity >95%) and [3H]-histamine (14.2 Ci·mmol−1) were from Hartmann Analytic (Braunschweig, Germany). All other reagents were from standard suppliers and of the highest purity available.

For the construction of the cDNA for hH4R-F169V, hH4R-S179A/M, hH4R-F169V+S179A/M, mH4R-V171F and mH4R-V171F+M181S, SDS-PAGE, Western blotting and cell culture, compare with Supporting Information. Cell culture, the generation of recombinant baculoviruses and membrane preparation (Gether et al., 1995; Schneider et al., 2009; Brunskole et al., 2011) as well as H4 receptor binding studies with [3H]-histamine and [35S]-GTPγS assays (Geyer and Buschauer, 2011; Geyer et al., 2014) were essentially performed as previously described with minor modifications (cf. Supporting Information).

The drug/molecular target nomenclature conforms to BJP's Concise Guide to Pharmacology (Alexander et al., 2013).

Results

Expression of recombinant proteins

Histamine H4 receptor wild types (hH4R, mH4R and rH4R) as well as mutants (hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M, hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S) were expressed in Sf9 insect cells together with G-protein subunits Gαi2 and Gβ1γ2 (Schneider et al., 2010). High expression at comparable ratios of both, receptors (wild types and mutants) and G-proteins, was confirmed by SDS-PAGE with Coomassie staining and densitometric analysis referred to the bands with apparent molecular weights of 78, 76, 33 and 30 kDa, respectively, present in all samples including the negative control (cf. Supporting Information Fig.  S1A and B), Western blots using anti-FLAG M1 and anti Gαi1/2 antibodies identified bands at 39 and 71 kDa, probably representing the unglycosylated and the glycosylated or the dimeric form of the receptor, as exemplarily shown for hH4R-F169V in Supporting Information Fig. S2. The Gαi2-protein appeared at 41 kDa (Supporting Information Fig. S2). Regardless of the high expression of the mH4 receptor, the rH4 receptor and the mH4R mutants, in these cases almost no specific binding of [3H]-histamine was detectable, which is in agreement with reported data for the mH4 receptor and rH4 receptor (Schnell et al., 2011), most probably due to the low affinity of histamine to these receptor proteins. Therefore, competition binding experiments with [3H]-histamine were not feasible at mH4 receptors, mH4 receptor mutants and rH4 receptors.

By contrast, high specific binding of [3H]-histamine to the hH4 receptors, hH4R-F169V, hH4R-S179A, hH4R-S179M mutant and to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants was detected. Bmax values ranged from 1.5 to 2.3 pmol [3H]-histamine mg−1 of soluble membrane protein and the Kd values of [3H]-histamine from 11.2 to 36.6 nM (Table 1 and Supporting Information Fig. S3).

Table 1.

Saturation binding data for [3H]-histamine at H4 receptor wild types and mutants

Receptor Kd (nM) Bmax (pmol·mg−1) n
hH4R-wt 11.16 ± 1.92 1.93 ± 0.32 3
hH4R-F169V 20.15 ± 4.47 1.92 ± 0.23 3
hH4R-S179M 17.81 ± 3.26 2.08 ± 0.02 3
hH4R-F169V+S179M 36.59 ± 4.24 1.52 ± 0.07 3
hH4R-S179A 14.81 ± 3.84 2.25 ± 0.16 3
hH4R-F169V+S179A 28.65 ± 3.57 1.46 ± 0.09 3
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Kd and Bmax values are given as mean ± SEM for n independent experiments, each performed in triplicate. Non-specific binding, amounting to 6.4–16.0% of total binding at 100 nM [3H]-histamine, was determined in the presence of 10 μM unlabelled histamine. The respective binding curves are available as Supplementary Material (Supporting InformationFig. S3).

[3H]-histamine competition binding experiments

The affinity at the hH4R-F169V mutant was in the same range or lower compared to the data at the wild-type hH4 receptor (Table 2). The decrease in affinity was pronounced for UR-PI376 (pKi 6.33 vs. 7.27), clozapine (pKi 5.51 vs. 6.18), isoloxapine (pKi 6.05 vs. 6.93) and clobenpropit (pKi 7.21 vs. 7.73). Effects of a single S179A or S179M mutation on affinity were marginal for most compounds, but higher affinity at hH4R-S179A compared to the wild type was determined in case of thioperamide, JNJ7777120, clozapine, isoloxapine and UR-PI376. At the double mutants, clozapine, isoloxapine and UR-PI376 showed reduced affinity, whereas the affinity of thioperamide and JNJ7777120 for the hH4R-F169V+S179A variant was even higher than for the hH4 receptor. In general, the pKi values were higher at the hH4R-S179A than at the hH4R-S179M single mutants, and higher at the hH4R-F169V+S179A than at the hH4R-F169V+S179M double mutants (Table 2).

Table 2.

[35S]-GTPγS and [3H]-histamine binding on hH4 receptor wild type and mutantsa

Ligand hH4R hH4R-F169V hH4R-S179M hH4R-F169V+S179M hH4R-S179A hH4R-F169V+S179A
Histamine pEC50 8.13 ± 0.06°° 7.72 ± 0.07**,°° 7.48 ± 0.08**,°° 7.24 ± 0.02**,°° 7.50 ± 0.05**,°° 7.36 ± 0.07**,°°
α 1 1 1 1 1 1
pKi 7.89 ± 0.04 7.59 ± 0.05* 7.49 ± 0.03** 7.40 ± 0.06** 7.61 ± 0.07* 7.45 ± 0.07**
UR-PI294 pEC50 8.35 ± 0.04°° 8.00 ± 0.11°° 7.98 ± 0.11°° 7.82 ± 0.02**,°° 8.16 ± 0.04°° 7.84 ± 0.01**,°°
α 1.02 ± 0.03 1.00 ± 0.07 0.98 ± 0.00 0.94 ± 0.05 0.92 ± 0.03 0.86 ± 0.08
pKi 7.84 ± 0.03 7.83 ± 0.04 7.93 ± 0.16 7.81 ± 0.05 7.90 ± 0.09 7.72 ± 0.08
Thioperamide pEC50 6.58 ± 0.06°° 6.52 ± 0.05°° 6.51 ± 0.04°° 6.60 ± 0.05°° 6.78 ± 0.06 7.28 ± 0.11
Α −1.39 ± 0.08°° −0.63 ± 0.06**,°° −1.19 ± 0.06°° −0.28 ± 0.04** −1.12 ± 0.06*,°° −0.23 ± 0.03**
pKb 6.83 ± 0.05 – – 6.81 ± 0.07 – 7.60 ± 0.10**,°°
pKi 6.75 ± 0.07 6.98 ± 0.15 6.67 ± 0.04 6.58 ± 0.06 7.34 ± 0.14* 7.29 ± 0.16
JNJ7777120 pEC50 7.10 ± 0.08°° 6.21 ± 0.12** 7.12 ± 0.03°° 7.28 ± 0.11 7.99 ± 0.08**,°° n.a.
Α −0.39 ± 0.03°° 0.43 ± 0.03** −0.48 ± 0.03°° 0.18 ± 0.04**,°° −0.66 ± 0.06**,°° 0**,°°
pKb 7.60 ± 0.05 – – 6.85 ± 0.16°° – 7.47 ± 0.09°°
pKi 7.16 ± 0.05 6.83 ± 0.05** 7.23 ± 0.07 6.81 ± 0.02** 7.78 ± 0.02** 7.48 ± 0.04*
Clozapine pEC50 6.24 ± 0.10°° 5.68 ± 0.12*,°° 6.26 ± 0.12°° 5.25 ± 0.04** 6.59 ± 0.10°° 5.71 ± 0.07*,°°
α 0.67 ± 0.04°° 0.56 ± 0.03°° 0.49 ± 0.08°° 0.49 ± 0.03°° 0.62 ± 0.09°° 0.36 ± 0.02**,°°
pKi 6.18 ± 0.03 5.51 ± 0.16* 6.36 ± 0.12 5.23 ± 0.14** 6.59 ± 0.11 5.48 ± 0.04*
Isoloxapine pEC50 7.08 ± 0.13°° 6.36 ± 0.10**,°° 7.26 ± 0.08°° 6.24 ± 0.09**,°° 7.36 ± 0.07°° 6.69 ± 0.03°°
α 0.81 ± 0.03°° 0.85 ± 0.09°° 0.62 ± 0.03°° 0.90 ± 0.03°° 0.77 ± 0.06°° 0.83 ± 0.10°°
pKi 6.93 ± 0.02 6.05 ± 0.13** 7.02 ± 0.10 6.24 ± 0.08** 7.47 ± 0.08* 6.68 ± 0.09
Clobenpropit pEC50 7.65 ± 0.11°° 7.63 ± 0.15°° 6.10 ± 0.15** n.a. n.a. n.a.
α 0.45 ± 0.04°° 0.27 ± 0.05* −0.44 ± 0.04**,°° 0**,° 0**,° 0**,°
pKb – – – 7.06 ± 0.07 7.42 ± 0.08 7.56 ± 0.16°
pKi 7.73 ± 0.07 7.21 ± 0.03** 7.14 ± 0.09** 7.23 ± 0.04** 7.56 ± 0.06 7.22 ± 0.02**
UR-PI376 pEC50 7.79 ± 0.08°° 6.25 ± 0.11** 6.93 ± 0.06**,°° 7.23 ± 0.12 7.28 ± 0.04°° 6.88 ± 0.18**,°°
α 1.11 ± 0.08°° 0.49 ± 0.02**,°° 0.80 ± 0.04**,°° 0.12 ± 0.01** 1.02 ± 0.06°° 0.25 ± 0.01**,°°
pKb – – – 5.82 ± 0.14** – 6.31 ± 0.22
pKi 7.27 ± 0.07 6.33 ± 0.11** 7.10 ± 0.12 6.18 ± 0.06** 7.60 ± 0.04 6.40 ± 0.07**
VUF8430 pEC50 7.42 ± 0.12°° 7.61 ± 0.07°° 7.41 ± 0.08°° 7.06 ± 0.13°° 7.53 ± 0.09°° 7.36 ± 0.09°°
α 0.84 ± 0.06 0.91 ± 0.06 0.85 ± 0.03 0.86 ± 0.01 0.85 ± 0.05 0.75 ± 0.06
pKi 7.84 ± 0.03 7.44 ± 0.02 7.55 ± 0.07 7.42 ± 0.15 7.81 ± 0.14 7.69 ± 0.15
Immepip pEC50 7.67 ± 0.05°° 7.73 ± 0.19°° 7.45 ± 0.10°° 7.45 ± 0.10°° 7.67 ± 0.09°° 7.68 ± 0.11°°
α 0.81 ± 0.03 0.85 ± 0.05 0.84 ± 0.09 0.84 ± 0.03 0.85 ± 0.06 0.65 ± 0.08
pKi 7.73 ± 0.16 7.47 ± 0.00 7.49 ± 0.09 7.54 ± 0.13 7.44 ± 0.08 7.52 ± 0.08
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apEC50 values ([35S]-GTPγS agonist mode), pKb values ([35S]-GTPγS antagonist mode), pKi values ([3H]-histamine competition binding) and α (intrinsic activity, maximal effect relative to histamine = 1.0) are given as mean ± SEM of at least three (up to nine) independent experiments, performed in triplicate. Results of statistical tests (one-way anova and Bonferroni post hoc tests): significant differences with respect to hH4 receptor – *P ≤ 0.05, **P ≤ 0.01; significant differences with respect to mH4 receptor – °P ≤ 0.05, °°P ≤ 0.01. In case of neutral antagonism (−0.25 ≤ α ≤ 0.25), pKb values were considered for statistical analysis instead of pEC50 values. Maximal effect α = 0: neutral antagonism, n.d.: not determined, n.a.: pEC50, pKb or pKi not applicable from performed experiments. Functional data for hH4 receptor compared with Nordemann et al. (2013).

Functional analysis of wild-type and mutant H4 receptors in the [35S]-GTPγS assay

We determined potencies (pEC50) and maximal effects (α) as well as antagonist activities (pKb) at wild-type and mutated receptors in the [35S]-GTPγS assay using agonists and antagonists respectively (Figure 1, Tables 2 and 3). Amounts of [35S]-GTPγS bound were similar except for mH4R-V171F+M181S, mH4R-V171F, mH4 receptor and rH4 receptor (Figure 4A). To facilitate comparison of the ratio of agonism to inverse agonism at the H4 receptor orthologues and mutants, the changes in [35S]-GTPγS binding were expressed as relative values in Figure 4B. In this representation, the span between maximal increase in [35S]-GTPγS binding elicited by the full agonist histamine and maximal decrease induced by the inverse agonist thioperamide was set to 100%. [35S]-GTPγS binding in the absence of ligand (water control) was set to zero (Figure 4B). The inverse agonism of thioperamide reflects the extent of constitutive activity of the respective wild-type or mutated H4 receptor (Figure 4). The response to thioperamide decreased in the order: hH4 receptor > hH4R-S179M > hH4R-S179A > hH4R-F169V > hH4R-F169V+S179M > hH4R-F169V+S179A > mH4R-V171F+M181S > mH4R-V171F = rH4 receptor = mH4 receptor. Thus, the single mutation F169V significantly decreased the exceptionally high constitutive activity of the hH4 receptor, and the mutation of hH4R-F169 and S179 into the corresponding amino acids of the mH4 and rH4 receptors caused a further decrease. The single hH4R-S179A or S179M mutation did not reduce constitutive activity significantly. Accordingly, F169 alone and in concert with S179 contributed to the high constitutive activity of the hH4 receptor. The mH4 receptor and the rH4 receptor did not show constitutive activity under the same conditions; thioperamide behaved as a neutral antagonist in the [35S]-GTPγS assay. This was also the case for the mH4R-V171F mutant, and there was no significant increase in constitutive activity for the mH4R-V171F+M181S mutant. The higher the constitutive activity, the lower is the relative ‘residual’ receptor capacity for activation by agonists (Figure 4B). Thus, the relative maximal response to histamine increased in the order: hH4 receptor < hH4R-S179M < hH4R-S179A < hH4R-F169V < hH4R-F169V+S179M < hH4R-F169V+S179A < mH4R-V171F+M181S < mH4R-V171F = rH4 receptor = mH4 receptor.

Table 3.

[35S]-GTPγS binding at mH4 receptor and rH4 receptor wild types and mH4 receptor mutantsa

Ligand hH4R mH4R-V171F+M181S mH4R-V171F mH4R rH4R
Histamine pEC50 8.13 ± 0.06°° 5.87 ± 0.05**,°° 5.95 ± 0.08**,°° 5.17 ± 0.14** 4.28 ± 0.06**,°°
α 1 1 1 1 1
UR-PI294 pEC50 8.35 ± 0.04°° 6.95 ± 0.11**,°° 7.25 ± 0.02**,°° 6.10 ± 0.11** 5.48 ± 0.08**,°°
α 1.02 ± 0.03 0.94 ± 0.04 0.99 ± 0.09 0.95 ± 0.03 1.09 ± 0.03
Thioperamide pEC50 6.58 ± 0.06°° 7.11 ± 0.08 n.a. n.a. n.a.
α −1.39 ± 0.08°° −0.20 ± 0.03** 0** 0** 0**
pKb 6.83 ± 0.05 7.84 ± 0.04**,°° 7.73 ± 0.09**,°° 7.12 ± 0.09** 6.44 ± 0.09°°
JNJ7777120 pEC50 7.10 ± 0.08°° n.a. 6.93 ± 0.12°° 6.10 ± 0.07** 6.13 ± 0.14
α −0.39 ± 0.03°° 0**,°° 0.42 ± 0.03** 0.44 ± 0.02** 0.24 ± 0.01**,°°
pKb 7.60 ± 0.05 5.90 ± 0.03** – – 4.93 ± 0.16**,°°
Clozapine pEC50 6.24 ± 0.10°° 5.71 ± 0.16*,°° 5.35 ± 0.03** n.a. n.a.
α 0.67 ± 0.04°° 0.41 ± 0.08*,°° 0.45 ± 0.04°° 0** 0**
pKb – – – 4.92 ± 0.04** 4.90 ± 0.09**
Isoloxapine pEC50 7.08 ± 0.13°° 6.01 ± 0.05**,°° 5.69 ± 0.16** n.a. 5.82 ± 0.16
α 0.81 ± 0.03°° 0.68 ± 0.05°° 0.44 ± 0.01*,° 0** 0.19 ± 0.03**
pKb – – – 5.26 ± 0.03** 5.12 ± 0.02**
Clobenpropit pEC50 7.65 ± 0.11°° 6.72 ± 0.13** 7.00 ± 0.15* 6.07 ± 0.09 n.a.
α 0.45 ± 0.04°° 0.35 ± 0.03 0.27 ± 0.04* 0.20 ± 0.02** 0**,°
pKb – – – 6.79 ± 0.00** 6.28 ± 0.04**
UR-PI376 pEC50 7.79 ± 0.08°° 6.08 ± 0.03** n.a. n.a. n.a.
α 1.11 ± 0.08°° 0.33 ± 0.04**,°° 0** 0** 0**
pKb – 6.08 ± 0.11 6.30 ± 0.10** 6.06 ± 0.17** 5.48 ± 0.03**
VUF8430 pEC50 7.42 ± 0.12°° 5.83 ± 0.16**,° 5.75 ± 0.18**,° 5.06 ± 0.14** 4.47 ± 0.15**
α 0.84 ± 0.06 0.73 ± 0.07 0.67 ± 0.05 0.68 ± 0.04 0.43 ± 0.05**
Immepip pEC50 7.67 ± 0.05°° 5.73 ± 0.06** 6.10 ± 0.12**,°° 5.27 ± 0.06** 4.95 ± 0.07**
α 0.81 ± 0.03 0.95 ± 0.03 0.66 ± 0.09 0.67 ± 0.08 0.68 ± 0.10
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a

For definition of symbols compare footnote with Table 2000; functional data for mH4 receptor and rH4 receptor compared with Nordemann et al. (2013).

Figure 4.

Figure 4

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Maximal agonistic effects of histamine and maximal inverse agonistic effects of thioperamide in [35S]-GTPγS assays. (A) Absolute values of bound [35S]-GTPγS (pmol·mg–1 protein) in the presence of histamine and thioperamide. Values demarcating light and dark grey bars represent the basal amount (in the absence of ligand) of bound [35S]-GTPγS. (B) For each H4 receptor species, the sum of the histamine and thioperamide effects was scaled to 100%; the zero line represents the ligand-free control. Significant changes: hH4R versus hH4R-F169V (P < 0.001), hH4R versus hH4R-F169V+S179A (P < 0.001), hH4R versus hH4RF169V+ S179M (P < 0.001), hH4R-F169V versus hH4R-F169V+S179A (P < 0.001), hH4R-F169V versus hH4RF169V+ S179M (P < 0.001) and mH4R versus mH4R-V171F+M181S (P < 0.05).

Concentration–response curves of histamine normalized to a percentual scale (maximal effect 100%) are shown in Figure 5A and Supporting Information Fig. S4A. The potency of histamine decreased from the hH4 receptor via hH4R-F169V, hH4R-S179A and hH4R-S179M mutants to the hH4 receptor double mutants by up to one order of magnitude (Table 2, Figure 5A and Supporting Information Fig. S4A). The potencies of histamine at the mH4 receptor and the rH4 receptor were low (pEC50 ∼ 4–5, Table 3, Figure 5A and Supporting Information Fig. S4A). Corresponding to the key role of F169 in the hH4 receptor, the potency was significantly higher at the mH4R-V171F and mH4R-V171F+M181S mutant than at the mH4 receptor wild type.

Figure 5.

Figure 5

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Concentration–response curves of eight ligands investigated in [35S]-GTPγS and [3H]-histamine competition binding assays. All curves are scaled with respect to a maximal histamine effect of 100%. Symbols and colours refer to the species variants and mutants respectively. Filled symbols: wild types; open symbols: mutants. (A) histamine; (B) UR-PI294; (C) thioperamide; (D) JNJ7777120; (E) clozapine; (F) VUF8430; (G) clobenpropit; (H) UR-PI376.

UR-PI294 (Igel et al., 2009b) was a full agonist with potencies being 5 to 10 times higher than those of histamine at all H4 receptor species variants (Figure 5B and Supporting Information Fig. S4B; Tables 2 and 3). The rank order at the hH4 receptor mutants corresponded to that of histamine. The pEC50 value at mH4R-V171F was in between the values at the hH4 receptor and mH4 receptor wild types, that is, the presence of F169, making the mH4 receptor more similar to the hH4 receptor, substantially increased the potency of UR-PI294, too.

The inverse agonistic response to thioperamide was highest at the hH4 receptor, slightly smaller at the hH4R-S179A (for concentration–response curves on this mutant, compare with Supporting Information Fig. S4C) and hH4R-S179M mutants, significantly reduced at the hH4R-F169V mutant and, in particular, at the double mutants, hH4R-F169V+S179A and hH4R-F169V+S179M (Figure 5C; Table 2). Whereas thioperamide acted as a weak partial inverse agonist at the mH4R-V171F+M181S mutant, it behaved as a neutral antagonist at the mH4 receptor, the rH4 receptor and the mH4R-V171F mutant with pKb values of 7.84, 7.12, 6.44 and 7.73 respectively.

JNJ7777120 was a partial inverse agonist at the highly constitutively active hH4 receptor and hH4R-S179A/M (Figure 5D and Supporting Information Fig. S4D; Tables 2 and 1995) but a partial agonist at the hH4R-F169V mutant, the mH4 receptor, the rH4 receptor and the mH4R-V171F mutant. At the double mutants as well as at the mH4R-V171F+M181S mutant, the compound rather behaved as a neutral antagonist.

Clozapine and isoloxapine were weak partial agonists or neutral antagonists at the mH4 receptor and the rH4 receptor (Figure 5E; Supporting Information Fig. S4E; Table 3). Introduction of phenylalanine into the mH4 receptor (mH4R-V171F mutant) significantly increased partial agonism of both compounds. Furthermore, at the hH4 receptor and its mutants, clozapine and isoloxapine acted as partial agonists. At the hH4R-F169V and the double mutants, the potencies were lower than at the wild-type receptor, with the maximal effects only decreasing in case of clozapine. In contrast, at the hH4R-S179M and S179A mutants, potencies of both clozapine and isoloxapine were similar to those at the hH4 receptor; maximal effects were reduced only at the S179M mutant. Generally, the potencies and the maximal effects of isoloxapine were higher than those of clozapine.

Both clobenpropit, a partial, and UR-PI376 (Igel et al., 2009a), a full agonist at the hH4 receptor, showed a considerable decrease in the maximal effects from the hH4 receptor wild type over the hH4R-F169V mutant to the double mutants, where clobenpropit revealed neutral antagonism (Figure 5G and H and Supporting Information Fig. S4G and H; Table 2). At the hH4R-S179M mutant, clobenpropit was a partial inverse agonist. At the hH4 receptor, the pEC50 values of UR-PI376 and clobenpropit were similar, whereas at the double mutants the pKb values of UR-PI376 were much lower than those of clobenpropit. At the mH4 receptor, the rH4 receptor, the mH4R-V171F and the mH4R-V171F+M181S mutant, both compounds behaved as weak partial agonists or neutral antagonists with maximal effects increasing from mH4 receptor over the mH4R-V171F to the mH4R-V171F+M181S mutants (Table 3).

The potent hH4 receptor agonists VUF8430 (Figures 5F; Supporting Information Fig. S4F; Tables 2 and 3) and immepip showed only little changes in pEC50 and α values at the five hH4 receptor mutants in comparison to the wild type. However, at the mH4 receptor, the rH4 receptor and the mH4R–V171F mutant, potencies and maximal effects were much lower.

Discussion and conclusions

Affinities and potencies of the investigated ligands at H4 receptor orthologues and mutants

Except for clobenpropit at hH4R-S179M, binding data were in the same range as the respective EC50 values from functional studies in the [35S]-GTPγS-assay (Table 2). Comparing mutant with wild-type receptors, changes in potency (Figure 6B and Supporting Information Fig. S5B) were higher than changes in affinity (Figure 6C and Supporting Information Fig. S5C), for example, in case of histamine and UR-PI294, indicating that the higher potencies of ligands at the hH4 receptor were a result of the higher constitutive activity. For most agonists, potencies were lower at hH4R-F169V and/or the double mutants than at the hH4 receptor, and higher at the mH4R-V171F and/or mH4R-V171F+M181S mutant than at the mH4 receptor (Tables 2 and 1995). Remarkable exceptions were VUF8430 and immepip with only minor effects of the F169V and the double mutations. With respect to histamine, clozapine and VUF8430, our results correlate with previous data (Lim et al., 2008), showing markedly reduced affinity for the hH4R-F169V compared to the wild type in the case of histamine and clozapine, whereas the affinity of VUF8430 was only slightly lowered.

Figure 6.

Figure 6

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Radar plots of maximal effects, potencies and affinities at wild type and three mutant human → mouse and two mouse → human H4 receptors. (A) Maximal effects (α values, relative to histamine = 1), (B) pEC50 values (or pKb in case of partial agonists with −0.25 ≤ α ≤ 0.25), (C) pKi values (n.a. for mH4R and mH4R mutants).

For clozapine and JNJ7777120, binding modes were proposed in which the phenyl and chlorophenyl moieties, respectively, occupy a pocket between TMs 3, 5, 6 and ECL2 (Lim et al., 2010; Kooistra et al., 2013). The phenyl rings of isoloxapine and UR-PI376 may adopt similar positions. For UR-PI294, clobenpropit, VUF8430 and immepip, the potencies at the hH4R-F169V mutant indicate no influence of F169 on binding. However, at the mH4R-V171F mutant these compounds are more potent than at the mH4 receptor wild type. The structures of these ligands suggest a binding mode different to that of JNJ7777120, clozapine and isoloxapine (pKi values: cf. Table 2). The potencies of histamine, JNJ7777120, clozapine, clobenpropit and UR-PI376 are different on at least one of the double mutants compared to the hH4R-F169V single mutant (Figure 6B). The additional mutation may either lead to a decrease in potency (histamine) or an increase (JNJ7777120) at both double mutants. The docking poses of histamine (Figure 3), clozapine and JNJ7777120 (Lim et al., 2010; Kooistra et al., 2013) do not indicate direct interactions with F169, but its substitution by valine may alter or destabilize the topology of the ligand binding pocket, in particular the conformation of L1755.39, L3266.58 and Y3407.35 (Figure 7) and, in turn, selectively affect ligand-receptor interactions. Alternatively or additionally, F169 at the entrance of the pocket may be part of the ‘optimal’ ligand binding path.

Figure 7.

Figure 7

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Intramolecular interactions specific for the hH4 receptor suggested from site-directed mutagenesis – F169V (mH4R, rH4R), S179M (mH4R) and S179A (rH4R) – and from an hH4 receptor model based on the crystal structure of the hH1 receptor. Colours of side chain atoms: N – blue, O – red. Interacting amino acids are represented as ball and stick model. Colours of carbons and backbone nitrogens: F169 and S179 – ochery; other amino acids different in the rH4 receptor and mH4 receptor – orange; further residues essential for interactions – cyan. A red dashed line indicates a hydrogen bond between S1795.43 and T3236.55. TMs are drawn as ribbons: TM5 – green, TM6 – light blue, TM7 – magenta. The C-terminal part of ECL2 and the N-terminal part of ECL3 are shown by tubes (cyan- and violet-coloured respectively).

In accordance with previous reports (Shin et al., 2002; Lim et al., 2008), the hH4R-S179A and S179M mutants suggest a minor role of S1795.43 on histamine binding. An increase in both potency and affinity (cf. thioperamide, JNJ7777120, clozapine, isoloxapine) due to S179A exchange may be interpreted as a hint that hydrophobic interactions come into play. For most ligands, pEC50 and pKi values are lower at hH4R-S179M than at hH4R-S179A (Table 2), possibly due to steric hindrance of ligand binding by the methionine side chain.

Different quality of action of JNJ7777120

The different degrees of constitutive activity of H4 receptor species orthologues become obvious from different qualities of action, inverse agonism, neutral antagonism or agonism of one and the same ligand. JNJ7777120 is a partial inverse agonist at the wild-type hH4 receptor, the hH4R-S179M and hH4R-S179A single mutants, and becomes a neutral antagonist at the double mutants hH4R-F169V+S179M and hH4R-F169V+S179A as well as at the mH4R-V171F+M181S mutant and a partial agonist at the mH4 receptor, the rH4 receptor and the mH4R-V171F mutant. Thus, JNJ7777120 fulfils the criteria of a protean agonist: inverse agonism at highly constitutively active receptors and partial agonism at lower or not constitutively active receptors (Kenakin, 2001). A striking exception is the hH4R-F169V mutant at which JNJ7777120 actually had to be expected to act as a weak partial inverse agonist, but showed partial agonism with similar potency as at the mH4 receptor and the rH4 receptor. Possibly, a ligand-specific stabilization of an active state due to the F169V exchange accounts for this apparent discrepancy. The chloro substituent in JNJ7777120 is suggested to interact with the side chain of hH4R-L1755.39 (Lim et al., 2010; Kooistra et al., 2013), which is close to F/V169 (Figure 7). These interactions within the JNJ7777120-occupied binding pocket may result in different qualities of action by stabilizing distinct conformations in wild-type and mutant receptors.

Maximal effects of agonists at H4 receptor orthologues and mutants

Among the investigated hH4 receptor agonists, histamine, UR-PI294, isoloxapine, VUF8430 and immepip do not show significantly reduced maximal effects at the hH4 receptor mutants compared to the wild-type receptor (Figure 6A and Supporting Information Fig. S5A). By contrast, in case of clozapine, clobenpropit and especially UR-PI376, decreasing maximal responses became obvious from the hH4 receptor over the F169V mutant to the double mutants. Except for UR-PI294 and immepip, which produced responses comparable to that of histamine at all tested H4 receptor species variants, the maximal agonistic effects (α values) were lowest at the mH4 receptor and the rH4 receptor (Figure 6A and Supporting Information Fig. S5A). A significant influence of the mH4R-V171F mutation was only observed with clozapine and isoloxapine. UR-PI376 was a partial agonist only at the mH4R-V171F+M181S mutant. Taking the different constitutive activities of the H4 receptor species variants into consideration, the situation becomes more complex in case of the agonists, too. Equal maximal effects at H4 receptor orthologues and mutants with high and low constitutive activities, respectively, result from different contributions to the stabilization of the active receptor state by one and the same agonist. Therefore, comparing maximal effects does not allow for drawing conclusions on selective impacts of F169 and/or S179 on receptor activation by different ligands. Furthermore, stabilization of an active state by agonists may be based on interactions different from those in the ligand-free, constitutively active receptor, that is, multiple active states must be taken into consideration. Therefore, beyond the G-protein activation used as readout in the present study, ligand-specific receptor conformations may trigger different signalling pathways according to the concept of functional selectivity [biased signalling; cf. conventional G-protein activation vs. β-arrestin recruitment (Rosethorne and Charlton, 2011; Nijmeijer et al., 2012)].

Constitutive activity

More than 40% of the GPCRs studied in vitro have been found to exhibit constitutive activity (Seifert and Wenzel-Seifert, 2002). Active GPCR states may be stabilized by intramolecular interactions in the ligand binding region, also in the absence of agonists. The key result of this study is the fact that the exceptionally high constitutive activity of the hH4 receptor is significantly reduced by the single F169V and the double F169V+S179M and F169V+S179A mutations, whereas the single S179M and S179A mutations do not significantly reduce constitutive activity. The effect of both amino acids, F169 (ECL2) and S1795.43, on the constitutive activity is cumulative. The mH4 receptor, the rH4 receptor and the mH4R-V171F mutant are not constitutively active. The constitutive activity is slightly increasing at the mH4R-V171F+M181S mutant. By contrast, high constitutive activity of the hH4 receptor is reflected by maximal inverse agonism of thioperamide, described as a full (Lim et al., 2005) or partial (Schneider et al., 2009) inverse agonist. In this context, the question arises whether thioperamide is a weaker partial inverse agonist at the hH4 receptor mutants than at the wild type or whether the maximal inverse agonistic effects only depend on different levels of constitutive activity. The assumption of comparable inverse agonism is supported by the fact that, at the hH4R-F169V and at the double mutants, but not at the hH4R-S179A and hH4R-S179M mutants, the minimum of [35S]-GTPγS binding in the presence of thioperamide approximately corresponds to that at the mH4 receptor and the rH4 receptor (Figure 4A). Moreover, in the case of the hH4 receptor and the double mutants, the pEC50, pKb and pKi values are similar (Table2, Figure 5). All criteria of constitutive activity (Seifert et al., 1998), high basal activity, high intrinsic activity and potency of partial agonists and a high inverse agonistic effect of inverse agonists, are fulfilled.

A possible explanation for the dependence of the high constitutive activity on the presence of F169 and S179 can be derived from a homology model of the hH4 receptor based on the crystal structure of the hH1 receptor (Shimamura et al., 2011). Our model indicates that F169 may adopt different conformations. Its phenyl ring may be directed towards the upper part of ECL2 like the corresponding tyrosine in the hH1 receptor or point to the ligand binding pocket. The first variant is rather unlikely due to an unfavourable polar environment and putative clashes with P166 (ECL2). In the second case shown in Figure 7, F169 is part of a hydrophobic cluster consisting of P166 (ECL2), L1755.39, L3266.58 and Y3407.35. Additionally, F169 contacts T333 (ECL3). A valine side chain as in the mH4 receptor, rH4 receptor and the hH4R-F169V mutants may interact only with P166 and/or L175. Furthermore, S1795.43 forms a hydrogen bond with T3236.55, which is impossible when S179 is exchanged by alanine or methionine as in the rH4R and the mH4R respectively. The cumulative effect on constitutive activity by mutation of both, F169 and S179, indicates that the agonist-free active state of the hH4 receptor is stabilized by hydrophobic interactions between ECL2 and the extracellular parts of TMs 5, 6 and 7 as well as the hydrogen bond between S1795.43 and T3236.55. In concert, these contacts favour a specific arrangement in particular of TMs 5 and 6, comparable to the stabilization of an active conformation by an agonist. An inward bulge of TM5 around position 5.46 and smaller inward movements of TMs 6 and 7 are characteristic of the activated β2-adrenoceptor compared to the inactive state (Rasmussen et al., 2011; Rosenbaum et al., 2011). At the cytoplasmic face of the receptor, an outward move of TM6 and rearrangements of TMs 5 and 7 are necessary for G-protein binding and contribute to the stabilization of active GPCR states. The TMs are suggested to behave as ‘oscillating arms’. When they move inwards at the extracellular side, they move outwards at the intracellular side and vice versa. Thus, the inward movement of TM5 and TM6 close to the agonist binding pocket results in an outward movement of these TMs at the ‘bottom’ of the receptor. In case of the hH4 receptor, a proximal arrangement of TMs 5 and 6 at the extracellular side becomes possible in the absence of bound agonist due to a network of interactions involving F169 and S179. However, also other amino acids contribute to the agonist-free stabilization of the active state of the hH4 receptor, since the double mutants still show a moderate degree of constitutive activity.

In case of the β2-adrenoceptor, an S204A+S207A double mutant showed about 50–60% lower constitutive activity than the β2-adrenoceptor wild type (Ambrosio et al., 2000). S2045.43 forms a hydrogen bond with N2936.55 (Rasmussen et al., 2011), corresponding to the suggested interaction of S1795.43 with T3236.55 in the hH4 receptor. A contribution of phenylalanine in ECL2 to constitutive activity by a network of hydrophobic interactions with amino acids in TMs 5, 6 and 7 has not been shown for other GPCRs, but may also play a role in other constitutively active receptors such as the hH3 receptor and the β2-adrenoceptor, which both contain the same FF motif as the hH4 receptor.

Conclusions

Until now, most studies on the constitutive activity of GPCRs have focused on the intracellular face, the DRY motif and the N-terminal part of TM6. The present study provides further evidence that intramolecular interactions in the agonist binding region contribute to the stabilization of ligand-free active GPCR states. Key result is the decrease in constitutive activity from the hH4 receptor over the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. Thus, F169 in ECL2 and S179 in TM5 play a major role in stabilizing a ligand-free active state of the hH4 receptor. Similar results on the β2-adrenoceptor suggest a common principle that may be of relevance for other GPCRs as well.

Acknowledgments

We are grateful to Dr Max Keller and Dr Tobias Holzammer for helpful discussions, to Susanne Gobleder for synthesizing and providing isoloxapine, to Maria Beer-Krön, Dita Fritsch and Gertraud Wilberg for expert technical assistance, and Dr Jens Schlossmann for giving access to his laboratory. This work was supported by the Graduate Training Programme (Graduiertenkolleg) GRK 760 and GRK 1910 of the Deutsche Forschungsgemeinschaft and by the European Cooperation in Science and Technology, COST Action BM0806, ‘Recent advances in histamine receptor H4R research’.

Glossary

ECL

extracellular loop

hH4R

human H4 receptor

ICL

intracellular loop

isoloxapine

8-chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine

mH4R

mouse H4 receptor

rH4R

rat H4 receptor

TM

transmembrane region

Author contributions

D. W., K. L., G. B., R. S. and A. B. conceived and designed the experiments. D. W., K. L. and U. N. performed the experiments. D. W., S. D. and A. S. performed computational chemistry. D. W., K. L., U. N., S. D., G. B., R. S. and A. B. analysed the data. D. W., G. B., R. S. and A. B. wrote the paper.

Conflict of interest

None.

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

Figure S1 Coomassie stained gels.

Figure S2 Western blots.

Figure S3 Saturation binding curves.

Figure S4 Concentration–response curves.

Figure S5 Radar plots.

bph0172-0785-sd1.pdf (978.9KB, pdf)

References

  1. Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters JA, Harmar AJ CGTP Collaborators. The Concise Guide to PHARMACOLOGY 2013/14: G protein-coupled receptors. Br J Pharmacol. 2013;170:1459–1562. doi: 10.1111/bph.12445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ambrosio C, Molinari P, Cotecchia S, Costa T. Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states. Mol Pharmacol. 2000;57:198–210. [PubMed] [Google Scholar]
  3. Ballesteros JA, Weinstein H. Integrated methods for the construction of three dimensional models and computational probing of structure function relations in G protein-coupled receptors. Method Neurosci. 1995;25:366–428. [Google Scholar]
  4. Brunskole I, Strasser A, Seifert R, Buschauer A. Role of the second and third extracellular loops of the histamine H(4) receptor in receptor activation. Naunyn Schmiedebergs Arch Pharmacol. 2011;384:301–317. doi: 10.1007/s00210-011-0673-3. [DOI] [PubMed] [Google Scholar]
  5. Buckland KF, Williams TJ, Conroy DM. Histamine induces cytoskeletal changes in human eosinophils via the H(4) receptor. Br J Pharmacol. 2003;140:1117–1127. doi: 10.1038/sj.bjp.0705530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng Y, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  7. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007;318:1258–1265. doi: 10.1126/science.1150577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KMJ, Ferguson DM, et al. A second generation force field for the simulation of proteins and nucleic acids. J Am Chem Soc. 1995;117:5179–5197. [Google Scholar]
  9. Dunford PJ, Holgate ST. The role of histamine in asthma. Adv Exp Med Biol. 2011;709:53–66. doi: 10.1007/978-1-4419-8056-4_6. [DOI] [PubMed] [Google Scholar]
  10. Dunford PJ, O'Donnell N, Riley JP, Williams KN, Karlsson L, Thurmond RL. The histamine H4 receptor mediates allergic airway inflammation by regulating the activation of CD4+ T cells. J Immunol. 2006;176:7062–7070. doi: 10.4049/jimmunol.176.11.7062. [DOI] [PubMed] [Google Scholar]
  11. de Esch IJ, Thurmond RL, Jongejan A, Leurs R. The histamine H4 receptor as a new therapeutic target for inflammation. Trends Pharmacol Sci. 2005;26:462–469. doi: 10.1016/j.tips.2005.07.002. [DOI] [PubMed] [Google Scholar]
  12. Gether U, Lin S, Kobilka BK. Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem. 1995;270:28268–28275. doi: 10.1074/jbc.270.47.28268. [DOI] [PubMed] [Google Scholar]
  13. Geyer R, Buschauer A. Synthesis and histamine H(3) and H(4) receptor activity of conformationally restricted cyanoguanidines related to UR-PI376. Arch Pharm. 2011;344:775–785. doi: 10.1002/ardp.201100144. [DOI] [PubMed] [Google Scholar]
  14. Geyer R, Kaske M, Baumeister P, Buschauer A. Synthesis and functional characterization of imbutamine analogs as histamine H3 and H4 receptor ligands. Arch Pharm. 2014;347:77–88. doi: 10.1002/ardp.201300316. [DOI] [PubMed] [Google Scholar]
  15. Igel P, Geyer R, Strasser A, Dove S, Seifert R, Buschauer A. Synthesis and structure-activity relationships of cyanoguanidine-type and structurally related histamine H4 receptor agonists. J Med Chem. 2009a;52:6297–6313. doi: 10.1021/jm900526h. [DOI] [PubMed] [Google Scholar]
  16. Igel P, Schneider E, Schnell D, Elz S, Seifert R, Buschauer A. N(G)-acylated imidazolylpropylguanidines as potent histamine H4 receptor agonists: selectivity by variation of the N(G)-substituent. J Med Chem. 2009b;52:2623–2627. doi: 10.1021/jm9000693. [DOI] [PubMed] [Google Scholar]
  17. Igel P, Dove S, Buschauer A. Histamine H4 receptor agonists. Bioorg Med Chem Lett. 2010;20:7191–7199. doi: 10.1016/j.bmcl.2010.10.041. [DOI] [PubMed] [Google Scholar]
  18. Jablonowski JA, Grice CA, Chai W, Dvorak CA, Venable JD, Kwok AK, et al. The first potent and selective non-imidazole human histamine H4 receptor antagonists. J Med Chem. 2003;46:3957–3960. doi: 10.1021/jm0341047. [DOI] [PubMed] [Google Scholar]
  19. Kenakin T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J. 2001;15:598–611. doi: 10.1096/fj.00-0438rev. [DOI] [PubMed] [Google Scholar]
  20. Kooistra AJ, Kuhne S, de Esch IJ, Leurs R, de Graaf C. A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design. Br J Pharmacol. 2013;170:101–126. doi: 10.1111/bph.12248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lange JHM, Wals HC, Vandenhoogenband A, Vandekuilen A, Denhartog JAJ. 2 novel syntheses of the histamine H-3 antagonist thioperamide. Tetrahedron. 1995;51:13447–13454. [Google Scholar]
  22. Leurs R, Chazot PL, Shenton FC, Lim HD, de Esch IJ. Molecular and biochemical pharmacology of the histamine H4 receptor. Br J Pharmacol. 2009;157:14–23. doi: 10.1111/j.1476-5381.2009.00250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lim HD, van Rijn RM, Ling P, Bakker RA, Thurmond RL, Leurs R. Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist. J Pharmacol Exp Ther. 2005;314:1310–1321. doi: 10.1124/jpet.105.087965. [DOI] [PubMed] [Google Scholar]
  24. Lim HD, Smits RA, Bakker RA, van Dam CME, de Esch IJP, Leurs R. Discovery of S-(2-Guanidylethyl)-isothiourea (VUF 8430) as a potent nonimidazole histamine H4 receptor agonist. J Med Chem. 2006;49:6650–6651. doi: 10.1021/jm060880d. [DOI] [PubMed] [Google Scholar]
  25. Lim HD, Jongejan A, Bakker RA, Haaksma E, de Esch IJ, Leurs R. Phenylalanine 169 in the second extracellular loop of the human histamine H4 receptor is responsible for the difference in agonist binding between human and mouse H4 receptors. J Pharmacol Exp Ther. 2008;327:88–96. doi: 10.1124/jpet.108.140343. [DOI] [PubMed] [Google Scholar]
  26. Lim HD, de Graaf C, Jiang W, Sadek P, McGovern PM, Istyastono EP, et al. Molecular determinants of ligand binding to H4R species variants. Mol Pharmacol. 2010;77:734–743. doi: 10.1124/mol.109.063040. [DOI] [PubMed] [Google Scholar]
  27. Ling P, Ngo K, Nguyen S, Thurmond RL, Edwards JP, Karlsson L, et al. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation. Br J Pharmacol. 2004;142:161–171. doi: 10.1038/sj.bjp.0705729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu C, Ma X-J, Jiang X, Wilson SJ, Hofstra CL, Blevitt J, et al. Cloning and pharmacological characterization of a fourth histamine receptor (H4) expressed in bone marrow. Mol Pharmacol. 2001a;59:420–426. doi: 10.1124/mol.59.3.420. [DOI] [PubMed] [Google Scholar]
  29. Liu C, Wilson SJ, Kuei C, Lovenberg TW. Comparison of human, mouse, rat, and guinea pig histamine H4 receptors reveals substantial pharmacological species variation. J Pharmacol Exp Ther. 2001b;299:121–130. [PubMed] [Google Scholar]
  30. Marson CM. Targeting the histamine H4 receptor. Chem Rev. 2011;111:7121–7156. doi: 10.1021/cr900166w. [DOI] [PubMed] [Google Scholar]
  31. Morse KL, Behan J, Laz TM, West RE, Jr, Greenfeder SA, Anthes JC, et al. Cloning and characterization of a novel human histamine receptor. J Pharmacol Exp Ther. 2001;296:1058–1066. [PubMed] [Google Scholar]
  32. Nakamura T, Itadani H, Hidaka Y, Ohta M, Tanaka K. Molecular cloning and characterization of a new human histamine receptor, HH4R. Biochem Biophys Res Commun. 2000;279:615–620. doi: 10.1006/bbrc.2000.4008. [DOI] [PubMed] [Google Scholar]
  33. Nguyen T, Shapiro DA, George SR, Setola V, Lee DK, Cheng R, et al. Discovery of a novel member of the histamine receptor family. Mol Pharmacol. 2001;59:427–433. doi: 10.1124/mol.59.3.427. [DOI] [PubMed] [Google Scholar]
  34. Nijmeijer S, Vischer HF, Rosethorne EM, Charlton SJ, Leurs R. Analysis of multiple histamine H4 receptor compound classes uncovers galphai and beta-arrestin2 biased ligands. Mol Pharmacol. 2012;82:1174–1182. doi: 10.1124/mol.112.080911. [DOI] [PubMed] [Google Scholar]
  35. Nordemann U, Wifling D, Schnell D, Bernhardt G, Stark H, Seifert R, et al. Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts. PLoS ONE. 2013;8:e73961. doi: 10.1371/journal.pone.0073961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Oda T, Matsumoto S. Identification and characterization of histamine H4 receptor. Nippon Yakurigaku Zasshi. 2001;118:36–42. doi: 10.1254/fpj.118.36. [DOI] [PubMed] [Google Scholar]
  37. O'Reilly M, Alpert R, Jenkinson S, Gladue RP, Foo S, Trim S, et al. Identification of a histamine H4 receptor on human eosinophils–role in eosinophil chemotaxis. J Recept Signal Transduct Res. 2002;22:431–448. doi: 10.1081/rrs-120014612. [DOI] [PubMed] [Google Scholar]
  38. Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP, Davenport AP, McGrath JC, Peters JA, Southan C, Spedding M, Yu W, Harmar AJ NC-IUPHAR. The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands. Nucl. Acids Res. 2014;42:D1098–D1106. doi: 10.1093/nar/gkt1143. (Database Issue): [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature. 2011;469:175–180. doi: 10.1038/nature09648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reher TM, Buschauer A, Neumann D, Seifert R. Incomplete activation of human eosinophils via the histamine H4-receptor: evidence for ligand-specific receptor conformations. Biochem Pharmacol. 2012;84:192–203. doi: 10.1016/j.bcp.2012.04.004. [DOI] [PubMed] [Google Scholar]
  41. Rosenbaum DM, Zhang C, Lyons JA, Holl R, Aragao D, Arlow DH, et al. Structure and function of an irreversible agonist-beta(2) adrenoceptor complex. Nature. 2011;469:236–240. doi: 10.1038/nature09665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosethorne EM, Charlton SJ. Agonist-biased signaling at the histamine H4 receptor: JNJ7777120 recruits beta-arrestin without activating G proteins. Mol Pharmacol. 2011;79:749–757. doi: 10.1124/mol.110.068395. [DOI] [PubMed] [Google Scholar]
  43. Rossbach K, Wendorff S, Sander K, Stark H, Gutzmer R, Werfel T, et al. Histamine H4 receptor antagonism reduces hapten-induced scratching behaviour but not inflammation. Exp Dermatol. 2009;18:57–63. doi: 10.1111/j.1600-0625.2008.00762.x. [DOI] [PubMed] [Google Scholar]
  44. Schmutz J, Kuenzle G, Hunziker F, Gauch R. Heterocycles with 7-membered rings. IX. 11- Amino substituted dibenzo[b,f]-1,4-thiazepines and -oxazepines. Helv Chim Acta. 1967;50:245–254. [Google Scholar]
  45. Schneider EH, Schnell D, Papa D, Seifert R. High constitutive activity and a G-protein-independent high-affinity state of the human histamine H(4)-receptor. Biochemistry. 2009;48:1424–1438. doi: 10.1021/bi802050d. [DOI] [PubMed] [Google Scholar]
  46. Schneider EH, Schnell D, Strasser A, Dove S, Seifert R. Impact of the DRY motif and the missing ‘ionic lock’ on constitutive activity and G-protein coupling of the human histamine H4 receptor. J Pharmacol Exp Ther. 2010;333:382–392. doi: 10.1124/jpet.109.163220. [DOI] [PubMed] [Google Scholar]
  47. Schnell D, Brunskole I, Ladova K, Schneider EH, Igel P, Dove S, et al. Expression and functional properties of canine, rat, and murine histamine H(4) receptors in Sf9 insect cells. Naunyn Schmiedebergs Arch Pharmacol. 2011;383:457–470. doi: 10.1007/s00210-011-0612-3. [DOI] [PubMed] [Google Scholar]
  48. Seifert R, Wenzel-Seifert K. Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol. 2002;366:381–416. doi: 10.1007/s00210-002-0588-0. [DOI] [PubMed] [Google Scholar]
  49. Seifert R, Wenzel-Seifert K, Lee TW, Gether U, Sanders-Bush E, Kobilka BK. Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor. J Biol Chem. 1998;273:5109–5116. [PubMed] [Google Scholar]
  50. Seifert R, Strasser A, Schneider EH, Neumann D, Dove S, Buschauer A. Molecular and cellular analysis of human histamine receptor subtypes. Trends Pharmacol Sci. 2013;34:33–58. doi: 10.1016/j.tips.2012.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, et al. Structure of the human histamine H-1 receptor complex with doxepin. Nature. 2011;475:65–70. doi: 10.1038/nature10236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shin N, Coates E, Murgolo NJ, Morse KL, Bayne M, Strader CD, et al. Molecular modeling and site-specific mutagenesis of the histamine-binding site of the histamine H4 receptor. Mol Pharmacol. 2002;62:38–47. doi: 10.1124/mol.62.1.38. [DOI] [PubMed] [Google Scholar]
  53. Smits RA, Lim HD, Stegink B, Bakker RA, de Esch IJ, Leurs R. Characterization of the histamine H4 receptor binding site. Part 1. Synthesis and pharmacological evaluation of dibenzodiazepine derivatives. J Med Chem. 2006;49:4512–4516. doi: 10.1021/jm051008s. [DOI] [PubMed] [Google Scholar]
  54. Strasser A, Wittmann HJ. Molecular modeling studies give hint for the existence of a symmetric hβ2R-Gαβγ-homodimer. J Mol Model. 2013;19:4443–4457. doi: 10.1007/s00894-013-1923-8. [DOI] [PubMed] [Google Scholar]
  55. Strasser A, Wittmann H-J, Buschauer A, Schneider EH, Seifert R. Species-dependent activities of GPCR ligands: lessons from histamine receptor orthologs. Trends Pharmacol Sci. 2013;34:13–32. doi: 10.1016/j.tips.2012.10.004. [DOI] [PubMed] [Google Scholar]
  56. Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov. 2008;7:41–53. doi: 10.1038/nrd2465. [DOI] [PubMed] [Google Scholar]
  57. Zampeli E, Tiligada E. The role of histamine H4 receptor in immune and inflammatory disorders. Br J Pharmacol. 2009;157:24–33. doi: 10.1111/j.1476-5381.2009.00151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhu Y, Michalovich D, Wu H-L, Tan KB, Dytko GM, Mannan IJ, et al. Cloning, Expression, and Pharmacological Characterization of a Novel Human Histamine Receptor. Mol Pharmacol. 2001;59:434–441. doi: 10.1124/mol.59.3.434. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Coomassie stained gels.

Figure S2 Western blots.

Figure S3 Saturation binding curves.

Figure S4 Concentration–response curves.

Figure S5 Radar plots.

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