Coupling of P2Y receptors to G proteins and other signaling pathways

Abstract

P2Y receptors are G protein-coupled receptors (GPCRs) that are activated by adenine and uridine nucleotides and nucleotide sugars. There are eight subtypes of P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄), which activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, differentiation, phagocytosis, secretion, nociception, cell adhesion, and cell migration. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, adenylyl and guanylyl cyclases, protein kinases, and phosphodiesterases. In addition, there are numerous ion channels, cell adhesion molecules, and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response.

Introduction

P2Y receptors belong to the superfamily of heptahelical receptors found exclusively in eukaryotes that operate by binding or sensing signals outside the cell and then activating intracellular processes by coupling to heterotrimeric G proteins. The main signaling molecules or agonists recognized by the eight subtypes of P2Y receptors are shown in Table 1 and consist of adenine and uridine nucleotides (ATP, ADP, UTP, and UDP) and nucleotide sugars (UDP-glucose). These nucleotides and nucleotide sugars are released basally from many cell types and release is intensified in response to mechanical stress, oxygen deprivation, viral infection, or apoptotic stimuli through opening of pannexin 1 channels.^1–4 In addition, ATP is stored in specialized secretory vesicles and released by exocytosis from nerve terminals, mast cells, chromaffin cells, and platelets. Platelets, in particular, have been shown to store ADP, ATP, UTP, and other factors in secretory vesicles called dense granules and release these stores upon platelet aggregation.⁵ The extracellular concentration of nucleotides is controlled by a family of ectoenzymes that catalyze the breakdown of nucleotides to nucleosides and by nucleoside transporters that transport nucleosides back into cells.^6,7 Besides the P2Y receptors, two other families of nucleoside/nucleotide receptors have been identified in eukaryotes: the P1 receptors (A₁, A_2A, A_2B, and A₃), which are G protein-coupled receptors activated by adenosine, and the P2X receptors (P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, and P2X₇), which are ligand-gated ion channels activated by ATP. These receptors (reviewed in Refs 8 and 9), are expressed throughout the body and have important roles in heart and immune functions and neurotransmission.^8,9

Table 1. P2Y Receptor Function and Coupling to G proteins.

Receptor	Agonist	G protein—Main Effector(s)	Function
P2Y1	ADP	Gq—PLCβ, Rac, Rho activation	Platelet shape change/aggregation Bone resorption Leptin secretion from adipocytes Mechanical and thermal nociception Atherosclerosis Angiogenesis Synaptic plasticity
P2Y2	ATP/UTP	Gq—PLCβ activation Go—PLCβ, Rac activation G12—Rho activation	Inhibition of bone formation and mineralization Immune cell recruitment and phagocytosis Vascular tone, inflammation, thrombosis Blood pressure regulation Intraocular pressure regulation Mechanical and thermal nociception HIV infection of T cells Epithelial K+/Cl− secretion Pancreatic and renal functions Liver regeneration Wound healing
P2Y4	UTP	Gq—PLCβ activation Go—PLCβ activation	Visual and auditory transmission Intestinal K+/Cl− secretion
P2Y6	UDP	Gq—PLCβ activation G12/13—Rho activation	Bone resorption Vascular tone and inflammation Cardiac fibrosis Macrophage cytokine/chemokine release Microglial phagocytosis Epithelial Cl− secretion
P2Y11	ATP	Gq—PLCβ activation Gs—AC activation	Inhibition of neutrophil apoptosis Negative regulation of TLR signaling Pancreatic Cl− secretion Cell cycle arrest in endothelium
P2Y12	ADP	Gi/o—AC inhibition PLCβ, RhoA activation	Platelet aggregation Microglial activation/migration Dendritic cell activation/macropinocytosis Peripheral anti-nociception, central nociception
P2Y13	ADP	Gi/o—AC inhibition PLCβ, RhoA activation	Bone formation Liver uptake of HDL Inhibition of ATP release from RBCs Peripheral anti-nociception
P2Y14	UDP/UDP-glucose	Gi/o—AC inhibition PLCβ activation	Immune function/IL-8 release in epithelium Gastric function/stomach contractility Peripheral anti-nociception

Data derived from Refs 3,11–67.

The general structure of P2Y receptors includes an extracellular N-terminus containing several potential N-linked glycosylation sites, seven transmembrane spanning domains that assist in forming the ligand binding pocket, three extracellular loops, three intracellular loops that participate in G protein coupling, and an intracellular C-terminus that contains several consensus binding/phosphorylation sites for protein kinases.¹⁰ Studies with selective P2Y receptor agonists and antagonists or mice lacking specific P2Y receptor subtypes have demonstrated an array of physiological functions coupled to these receptors,^3,11–67 which are summarized in Table 1.

P2Y Receptor Coupling to G Proteins

Heterotrimeric G proteins consist of a Gα subunit that is tightly associated with Gβγ subunits and bind to G protein-coupled receptors (GPCRs) at the inner surface of the cell. P2Y receptors, like other members of the GPCR superfamily, act as guanine nucleotide exchange factors (GEFs) for heterotrimeric G proteins, causing dissociation of the G protein from the activated receptor as well as dissociation of the Gα subunit from the Gβγ complex. These separated G protein subunits can then interact with a variety of effector proteins, leading to either activation or deactivation of the effector protein. On the basis of the sequence similarity of the Gα subunits, heterotrimeric G proteins have been broadly divided into four subfamilies (G_q, G_s, G_i/o, and G_12/13) that are used to define both receptor and effector coupling.⁴³ Similarly, the P2Y receptors have been grouped into two subfamilies based on their sequence similarity: the G_q-coupled P2Y receptors, which share a 28–52% sequence homology and include the P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors, and the G_i/o-coupled P2Y receptors, which share a 45–50% sequence homology and include the P2Y₁₂ P2Y₁₃, and P2Y₁₄ receptors. There are, however, anomalies to this classification in that most of the G_q-coupled P2Y receptors are known to link to other G protein subfamilies. These additional G protein linkages are shown in Table 1.

The ability of individual P2Y receptor subtypes to couple to specific G proteins was initially derived from indirect evidence obtained by measuring the cytoplasmic levels of second messengers, i.e., inositol phosphate, Ca²⁺ and cyclic AMP (cAMP), in response to nucleotide stimulation or by determining the sensitivity of nucleotide-induced signaling to the G_i/o protein inhibitor pertussis toxin (PTX). More recently, direct evidence for P2Y₁ receptor coupling to G_q proteins (Gα_qβ₁γ₂ and Gα₁₁β₁γ₂) was obtained by measuring agonist-induced GTP hydrolysis in vesicles reconstituted with these proteins.⁴⁴ Similarly, vesicle reconstitution of the P2Y₁₂ receptor with specific G proteins indicates that this receptor couples to Gα_i2 more effectively than to Gα_i1 and Gα_i3, but not to Gα_o or Gα_q.⁴⁵ Coupling of the P2Y₁ receptor to G_q results in the activation of PLCβ and mobilization of intracellular Ca²⁺ as well as the activation of the monomeric G proteins, RhoA and Rac,⁴⁶ while coupling of the P2Y₁₂ receptor to G_i2 results in the inhibition of adenylyl cyclase as well as the activation of PI3K, Akt, Rap1b, and potassium channels. Together, the P2Y₁ and P2Y₁₂ receptors, which are activated by ADP, control platelet shape change and aggregation and their effects have been extensively reviewed.^35,47

The P2Y₂ receptor is activated equally well by ATP or UTP and couples to G_q, G_o, and G₁₂ proteins, which was determined by measuring GTPγ [³⁵S] binding to specific Gα subunits in response to agonist stimulation in 1321N1 astrocytoma cells.^48,49 These studies also indicate that coupling of the P2Y₂ receptor to G_o and G₁₂ requires interaction with αv integrins via a three amino acid integrin-binding domain (RGD) in the first extracellular loop of the P2Y₂ receptor to access and activate G_o and G₁₂, whereas coupling to G_q does not require integrin interaction.^48–50 Activation of G_o by the P2Y₂ receptor leads to activation of RhoA, whereas activation of G₁₂ leads to activation Rac and the RacGEF, Vav2, which together are important for cytoskeletal rearrangements involved in cell migration and phagocytosis (see Figure 1). By using antisense oligonucleotides against specific G protein subunits, the P2Y₂ receptor in HEL cells was shown to activate PLCβ and mobilize intracellular Ca²⁺ by coupling to Gα₁₆, a member of the G_q subfamily⁵¹ and to activate PLCβ₁ and PLCβ₃ in gastric smooth muscle cells via coupling to Gα_q and the βγ subunits of Gα_i3β₁γ₂, respectively.⁵²

Figure 1.

Model of cytoskeletal signaling by the P2Y₂ receptor through G_o and G₁₂ proteins. The P2Y₂ receptor, which is linked to the actin cytoskeleton through association with filamin A,⁶⁷ initiates cytoskeletal rearrangements involved in cell migration, neurite extension and phagocytosis by promoting the growth of actin filaments near the activated receptor. This is accomplished, in part, by the interaction of the P2Y₂ receptor RGD domain with α_Vβ_3/5 integrins.⁵⁰ Integrin interaction allows the P2Y₂ receptor to access and activate G_o and G₁₂ proteins that are known to associate with β₃ integrins and with the integrin-associated protein, CD47.^49,50 Activation of G_o and G₁₂ leads to activation of Rac1 and RhoA, which then activate the actin-binding LIM kinases (LIMKs) responsible for serine-phosphorylation/deactivation of the actin disassembly protein, cofilin.^48,49

The P2Y₄ receptor is activated by UTP and couples to G_q and G_o proteins to activate PLCβ and mobilize intracellular Ca²⁺.^53,54 This receptor also activates Rho and inhibits K⁺ currents in myocytes from rat cerebral arteries in a Rho-dependent manner.⁵⁵

The P2Y₆ receptor is activated by UDP and couples to members of the G_q and G_12/13 subfamilies. In most cell types tested, the P2Y₆ receptor couples to G_q to activate PLCβ and mobilize intracellular Ca²⁺, whereas coupling to G_o appears to be absent.¹⁰ However, in rat sympathetic neurons injected with P2Y₆ receptor mRNA, PTX inhibits the decrease in voltage-gated Ca²⁺ currents mediated by this receptor, suggesting that the P2Y₆ receptor, in some instances, may operate through G_i/o.⁵⁶ Coupling of the P2Y₆ receptor to G_12/13 and Rho activation was recently demonstrated to contribute to pressure overload-induced cardiac fibrosis in an in vivo mouse model.⁵⁷ Nishida et al. generated mice with a cardiomyocyte-specific overexpression of p115-RGS, a regulator of the G protein signaling that specifically inhibits Gα_12/13, to show that these mice have suppressed expression of fibrogenic genes in cardiomyocytes in response to mechanical stretch that is dependent on Rho activation. The authors also demonstrated that the mechanism of mechanical stretch-induced fibrosis involves the release of ATP and UDP from cardiomyocytes through pannexin 1 channels that activate the P2Y₆ receptor.⁵⁷

The P2Y₁₁ receptor is activated by ATP and couples to members of the G_q and G_s subfamilies. G_s proteins stimulate adenylyl cyclase to increase production of cAMP and coupling of the P2Y₁₁ receptor to G_s is important for controlling several physiological processes.^36,58,59 In human monocytes, activation of the P2Y₁₁ receptor negatively regulates toll-like receptor signaling by increasing cAMP production.⁵⁸ Activation of P2Y₁₁ receptors inhibits neutrophil apoptosis by a cAMP-dependent mechanism³⁶ and activates cAMP-dependent Cl⁻ channels on the baso-lateral surface of pancreatic duct cells.⁵⁹ Although ATP appears to be the main agonist for the P2Y₁₁ receptor, UTP has been reported to stimulate Ca²⁺ mobilization in P2Y₁₁ receptor-transfected 1321N1 astrocytoma cells by a PTX-sensitive mechanism, whereas Ca²⁺ mobilization induced by ATP in these cells is insensitive to PTX, suggesting that the P2Y₁₁ receptor is capable, in some instances, of coupling to G_o in addition to G_q.⁶⁰ It is uncertain why the agonist profile and G protein coupling of the P2Y₁₁ receptor is altered in this cell expression system, although it is possible that these differences may be due to the ability of the P2Y₁₁ receptor to heterodimerize with other GPCRs. In support of this possibility, co-expression of the P2Y₁ and P2Y₁₁ receptors in HEK cells was found to alter the agonist profile from that in cells expressing only the P2Y₁₁ receptor.⁶¹

The P2Y₁₂ receptor is activated by ADP and couples mainly to G_i, although G_o coupling is indicated in some cell types, as is PTX-insensitive signaling by the P2Y₁₂ receptor.⁶² Soulet et al.⁶² have shown that expression of the P2Y₁₂ receptor in CHO cells mediates PTX-sensitive cell proliferation that relies on signaling through PI3-kinase/Akt and ERK1/2, and PTX-insensitive actin cytoskeleton organization that relies on signaling through RhoA/Rho-kinase. Conversely, in C6 glioma cells, activation of the P2Y12 receptor enhances cell proliferation by a pathway that is dependent on Gα_i, RhoA/ROCK, and PKCζ, but does not involve ERK1/2 signaling.⁶³ In mouse dendritic cells, where the P2Y₁₂ receptor is endogenously expressed, agonist stimulation of the P2Y₁₂ receptor causes a PTX-sensitive increase in cytoplasmic Ca²⁺ that is necessary for macropinocytosis of antigens,¹² suggesting that the P2Y₁₂ receptor in dendritic cells can couple to G_o.

The P2Y₁₃ receptor couples to G_i/o and is activated by ADP. In transfected 1321N1 or CHO cells, agonist-induced activation of the P2Y₁₃ receptor increases the PTX-sensitive production of IP₃ and inhibits forskolin-mediated cAMP production, whereas high concentrations of P2Y₁₃ receptor agonists cause an increase in cAMP production, suggesting that the P2Y₁₃ receptor can, in some instances, couple to G_s.⁶⁴ In human hepatocytes, endocytosis of HDL mediated by the P2Y₁₃ receptor requires the activation of RhoA and ROCK I, although the subtype of G protein involved in this process was not confirmed.⁶⁵ Similarly, the P2Y₁₃ receptor activates RhoA and ROCK I in mouse osteoblasts and plays an important role in bone formation.¹⁷ In red blood cells, Wang et al.⁴⁰ demonstrate that activation of the P2Y₁₃ receptor decreases the release of ATP by these cells, by inhibiting the production of cAMP.

The P2Y₁₄ receptor couples to G_i/o and is activated by UDP, UDP-glucose, and other UDP-linked sugars, including UDP-galactose and UDP-N-acetylglucosamine. The signaling properties of this receptor were recently reviewed⁶⁶ and its physiological functions are beginning to be revealed. Studies in mouse and human tissues indicate that the P2Y₁₄ receptor is highly expressed in mucosal epithelium where its activation leads to the production of chemokines and cytokines involved in the recruitment of neutrophils, including IL-8, macrophage inflammatory protein-2, and keratinocyte-derived cytokine.³⁸ Also, high levels of the P2Y₁₄ receptor are expressed in the rat forestomach where it plays a role in muscle contractility.¹³

Other Mechanisms of P2Y Receptor Signaling

P2Y Receptor Oligomerization

It is generally accepted that GPCRs can dimerize to form heteromeric and homomeric structures or more complex oligomeric structures and, accordingly, several examples of dimerization involving P2Y receptors have been characterized by performing co-immunoprecipitation experiments and/or FRET analysis. Both the P2Y₁ and P2Y₂ receptors form a heteromeric complex with the A₁ adenosine receptor in HEK293 cells.^68,69 Heteromeric association between the G_i-coupled A₁ receptor and the G_q-coupled P2Y₁ receptor was shown to create a hybrid receptor that responds predominantly to the P2Y₁ receptor agonist ADPβS to activate both G_i and G_q.⁶⁹ Specifically, activation of G_i and inhibition of forskolin-induced cAMP accumulation by the A₁-P2Y₁ receptor complex displays a hybrid selectivity for P2Y₁ and A₁ receptor agonists and antagonists, whereas activation of G_q and generation of inositol phosphates occurs only with P2Y₁ receptor agonists and not with A₁ receptor agonists,⁶⁹ suggesting that the cross talk to G proteins for the A₁–P2Y₁ receptor complex is unidirectional. In contrast, association of the A₁ receptor with the P2Y₂ receptor does not affect the ligand selectivity of the heteromeric receptor, although simultaneous stimulation of the A₁–P2Y₂ receptor complex with A₁ and P2Y₂ receptor agonists interferes with G_i signaling and enhances G_q signaling.⁶⁸

The P2Y₁ receptor also is thought to form a heteromeric complex with the P2Y₁₁ and P2Y₁₂ receptor,^61,70 which for the P2Y₁₁ receptor alters its function. Ecke et al. have shown that co-expression of the P2Y₁ and P2Y₁₁ receptors in HEK293 or 1321N1 cells promotes agonist-induced endocytosis of the P2Y₁₁ receptor, which when expressed alone in these cells is unable to undergo agonist-induced endocytosis. The ligand selectivity of the P2Y₁–P2Y₁₁⁶¹ receptor complex, measured by activation of Ca²⁺ signaling, is also different than the agonist potency profile in cells expressing only the P2Y₁₁ receptor.

Recent publications show that the P2Y₄, P2Y₆, and P2Y₁₂ receptors form homo-oligomeric complexes that are thought to be involved in receptor signaling.^71,72 Importantly, Savi et al.⁷¹ have shown that clopidogrel, an antithrombotic agent that antagonizes the platelet P2Y₁₂ receptor, interferes with homo-oligomeric assembly of the P2Y₁₂ receptor and localization of these complexes into lipid rafts that is required for receptor activation. D'Ambrosi et al.⁷² have shown that in neuronal cells the P2Y₄ and P2Y₆ receptors form homo- and hetero-oligomeric complexes, that UTP affects the oligomerization of the P2Y₆ receptor, but not the P2Y₄ receptor, and that dimeric P2Y₄ receptors and monomeric P2Y₆ receptors selectively partition into lipid rafts from specialized subcellular compartments, such as synaptosomes.

P2Y Receptor Desensitization and Trafficking

Agonist-induced activation of GPCRs often initiates receptor desensitization, which diminishes GPCR responsiveness to ligands and can lead to receptor internalization or endocytosis. In addition, the kinetics of GPCR desensitization and recovery from desensitization was recently demonstrated to be required for sufficient activation of store-operated, calcium release-activated calcium (CRAC) channels that are tightly linked to expression of the c-fos transcripition factor, indicating that GPCR desensitization is also important for activating gene expression.⁷³ Agonist-dependent GPCR desensitization is generally mediated by a family of GPCR kinases (GRK 1-7), which phosphorylate serine/threonine residues in intracellular domains of the receptor and, in some instances, promote the binding of β-arrestins (β-arrestin1-4). β-arrestins, in turn, can regulate several receptor functions, including desensitization (by inhibiting GPCR/G-protein interaction), endocytosis (by interacting with clathrin and AP2 to promote receptor internalization into clathrin-coated pits) and signaling (by interacting with MAPKs and Src).⁷⁴ A recent study looked at P2Y_{1,2,4,6,11,12} receptor interactions with β-arrestins in transfected HEK-293 cells using FRET analysis or by measuring β-arrestin translocation to the plasma membrane in response to receptor activation.⁷⁵ A summary of these interactions is shown in Table 2. In this study, Hoffmann et al. determined that all six of these P2Y receptor subtypes interact to some degree with β-arrestin2, whereas the P2Y₂ and P2Y₄ receptors interact with both β-arrestin1 and β-arrestin2. Interestingly, they found that the P2Y₂ receptor couples differently to β-arrestins depending on whether ATP or UTP is used to activate this receptor; activation of the P2Y₂ receptor with ATP causes a strong interaction with β-arrestin1 and a weak interaction with β-arrestin2, whereas activation of the P2Y₂ receptor with UTP causes a strong interaction with both β-arrestin1 and β-arrestin2. This differential coupling of the P2Y₂ receptor to β-arrestins in response to stimulation with ATP or UTP supports earlier findings indicating that desensitization of the P2Y₂ receptor requires 10-fold higher concentrations of ATP than UTP, even though these nucleotides activate P2Y₂ receptor signaling processes with similar potencies.⁷⁶

Table 2. P2Y Receptor Trafficking.

Receptor	Coupling to β-Arrestins	Mechanism of β-Arrestin Coupling	Mechanism of Internalization	Apical/Basolateral Expression in Polarized Cells	Mechanism of Coupling to the Cytoskeleton
P2Y1	β-arrestin2	PKC phosphorylation	Dynamin	Basolateral (dep. on basic/acidic aa ratio in the C-terminus)
P2Y2	β-arrestin2 (ATP) β-arrestin1 and 2 (UTP)	GRK2 phosphorylation	Dynamin-independent	Apical (RGD domain-dep.)	Filamin A
P2Y4	β-arrestin1 and 2	GRK phosphorylation	Dynamin	Apical
P2Y6	β-arrestin2	GRK2 phosphorylation	Dynamin	Apical
P2Y11	β-arrestin2	GRK2 phosphorylation	Dynamin, dimerization with P2Y1	Basolateral
P2Y12	β-arrestin2	GRK2,6 phosphorylation	Dynamin, PDZ domain-dep.	Basolateral
P2Y13	unknown			Unsorted
P2Y14	unknown			Basolateral

Data derived from Refs 67,75,77–85.

β-arrestins are, in part, responsible for GPCR-mediated activation of the MAPKs, ERK1/2, by inducing receptor endocytosis and relocalization to endosomes.⁷⁴ The P2Y₂ receptor, which displays differential coupling to β-arrestins in response to activation by ATP versus UTP, also displays a differential effect on the duration of ERK1/2 activation in response to these endogenous P2Y₂ receptor ligands (i.e., ATP causes sustained ERK1/2 activation, whereas UTP causes transient ERK1/2 activation).⁷⁵ In numerous studies, the duration of ERK1/2 activation is critical for determining cell fate (i.e., whether cells differentiate, proliferate, procreate or die).⁸⁶ This suggests that the P2Y₂ receptor may regulate different cell fates, depending on which receptor agonist is present.

In rat mesenteric smooth muscle cells, β-arrestin2 and GRK2 are required for P2Y₂ receptor desensitization and termination of p38 and ERK1/2 signaling.^77,78 Receptor mutagenesis studies indicate that deletion of structural motifs or point mutations of putative GRK phosphorylation sites (e.g., S²⁴³A, T³⁴⁴A, and S³⁵⁶A) in the P2Y₂ receptor C-terminal tail diminishes agonist-induced desensitization and internalization of this receptor.^79,80 Suppression of specific GRKs with siRNA demonstrate that desensitization of the P2Y₁₂ receptor requires GRK2 and GRK6 activity.⁸¹ In contrast, P2Y₁ receptor desensitization is largely dependent on PKC activity.⁸¹ In HEK293 cells, a dominant negative dynamin construct (dyn-K⁴⁴A) was used to demonstrate that agonist-induced internalization of the P2Y_1,4,6,11,12 receptors is dynamin-dependent, whereas agonist-induced internalization of the P2Y₂ receptor is dynamin-independent.⁷⁵

Activation of receptor tyrosine kinases by growth factors has been shown to resensitize P2Y receptors in many different cell types by PKC-dependent P2Y receptor phosphorylation.^87,88 In retinal glial cells, P2Y receptors are desensitized by ATP and resensitized by growth factors (e.g., EGF, PDGF, NGF), via activation of PI3K and protein phosphatases, whereas resensitization is not dependent on the activity of PKC, Src kinases, or ERK1/2.⁸⁹

Many GPCRs express C-terminal PDZ-binding domains (type I: X–S/T–X–O, type II: X–O–X–O, type III: X–D/E–X–O, where X = any and O = hydrophobic amino acid) that interact with PDZ domain-containing proteins and have established roles in receptor trafficking, localization, and assembly of signaling complexes.⁹⁰ Several P2Y receptors contain PDZ-binding domains, including the P2Y_1,2,4,12,14 receptors, and the PDZ-binding domain in the extreme C-terminal tail of the P2Y₁₂ receptor (ETPM) has been shown to be important for receptor trafficking in platelets.⁸² Disruption or removal of the P2Y₁₂ PDZ-binding domain blocks interaction with β-arrestin and slows receptor internalization in CHO cells, which the authors suggest is either due to interaction of the PDZ-binding domain with a regulatory kinase or phosphorylation of the threonine residue within the PDZ-binding domain that is required for β-arrestin interaction.⁸²

Polarized expression of P2Y receptors in apical versus basolateral surfaces of epithelial cells has been observed (see Table 2). Wolff et al.⁸³ demonstrated that the P2Y_1,11,12,14 receptors are basolaterally expressed, whereas the P2Y_2,4,6 receptors are apically expressed and the P2Y₁₃ receptor is unsorted in polarized Madin-Darby canine kidney (MDCK) cells. Mutagenesis studies with the P2Y₁ and P2Y₂ receptors indicate that charged amino acid residues (five basic and four acidic) in the C-terminal tail of the P2Y₁ receptor are important for basolateral trafficking,⁸⁴ whereas the RGD domain in the first extracellular loop of the P2Y₂ receptor is important for apical trafficking in polarized MDCK cells.⁸⁵

P2Y Receptor Cross Talk

Receptor cross talk refers to the simultaneous activation of different types of cell surface receptors resulting in coordinated stimulation or suppression of signal transduction. Cross talk with P2Y receptors can produce a synergistic or more than additive change in the level of second messengers. For example, co-activation of various G_i-coupled receptors and G_q-coupled P2Y receptors induces a synergistic rise in intracellular IP₃ and Ca²⁺ levels^91–94 and the release of arachidonic acid (AA)^93,95, as compared to activation of G_q-coupled P2Y receptors alone. Also, prestimulation of G_q-coupled P2Y₁ or P2Y₂ receptors in HEK293 cells enables the G_i-coupled CXCR2 chemokine receptor and the G_s-coupled β–adrenergic receptor to mobilize intracellular Ca²⁺, a response not normally regulated by these G_i- and G_s-coupled receptors.⁹⁴ G_q-coupled P2Y receptors stimulate PLC activity that generates DAG, an endogenous activator of PKC, which in turn can activate phospholipase A2 and generate AA from membrane phospholipids. Although activation of G_i-coupled m2 muscarinic, α₂ adrenergic, and D₂ dopaminergic receptors in CHO-K1 cells does not induce AA release, these receptor activities can enhance the effect of P2Y receptor activation on AA generation.⁹⁵ Activation of the P2Y₂ receptor resensitizes vanilloid type1 channels (TRPV1) in kidney sensory neurons⁹⁶ and activation of the P2Y₁ receptor causes PKC-dependent phosphorylation of the capsaicin receptor (a VR1 cation channel), which shifts the capsaicin concentration–response curve twofold to the left, and decreases the threshold for capsaicin receptor activation by heat from 42 to 35°C.⁹⁷ Thus, cross talk between the P2Y₁ and capsaicin receptors may represent a novel mechanism for the perception of pain (nociception) induced by P2Y receptor activation.

Cross talk between P2Y receptors and P2X receptor ion channels has also been demonstrated. In Xenopus oocytes expressing the recombinant human P2X₁ receptor, a transient inward current occurs in response to ATP.⁹⁸ This response undergoes desensitization (i.e., current flow decreases upon prolonged or repeated exposure to ATP), however, co-expression and co-activation of either the P2Y₁ or P2Y₂ receptor (stimulated by ADP or UTP, respectively) inhibits P2X₁ receptor desensitization. The mechanism of P2Y receptor-mediated inhibition of P2X₁ receptor desensitization does not appear to involve direct phosphorylation of the P2X₁ receptor, but does involve protein kinase activity perhaps mediated by an accessory protein.⁹⁸ P2X₁ receptors can also modulate P2Y receptor activity. In megakaryocytes, P2X₁ receptor activation with α,β-meATP causes a rapid and transient Ca²⁺ influx, whereas P2Y₁ receptor activation with ADP causes a slower, larger, and more sustained increase in cytoplasmic Ca²⁺.⁹⁹ Co-application of the two agonists, however, accelerates the Ca²⁺ response and potentiates the peak amplitude, suggesting that the P2X₁ receptor may have a priming role in the activation of P2Y₁ receptors during platelet stimulation. The P2Y₁₂ receptor, through activation of PI3K, also has been shown to play a synergistic role in the regulation of both P2Y₁ and P2X₁ receptor-mediated currents in megakaryocytes¹⁰⁰ and activation of P2Y₂ receptors resensitizes P2X₂ receptor channels in bladder sensory neurons.¹⁰¹

P2Y Receptor Coupling to Ion Channels

The generation of knock-out (KO) mouse models has helped to identify ion channels controlled by various subtypes of P2Y receptors, which have been extensively reviewed.^10,102,103 In respiratory epithelium, the generation of the P2Y₂ receptor KO mouse helped to show that the major luminal P2Y receptor involved in the activation of Cl⁻ secretion is the P2Y₂ receptor subtype. Not all epithelia express the P2Y₂ receptor subtype luminally, but still respond robustly to the addition of luminal UTP. Mouse jejunum displays luminal UTP-stimulated Cl⁻ secretion, which was not affected in P2Y₂ receptor KO mice.¹⁰⁴ Using a P2Y₄ receptor KO mouse, it was recently established that the P2Y₄ receptor is responsible for the UTP-stimulated Cl⁻ secretion.¹⁰⁵

The P2Y₁ receptor contains a PDZ-binding domain (DTSL) in its C-terminal tail that interacts directly with the Na⁺/H⁺-exchange regulatory factor (NHERF-2) to control Na⁺/H⁺ exchange.¹⁰⁶ Similarly, an RRSE-QXK/RSE motif that is present in P2Y_1,2,6,11 receptors is required for modulation of a voltage-gated ion channel that mediates a transient inward current in Xenopus oocytes.¹⁰⁷

Activation of the P2Y₂ receptor in many cell types causes the cytoplasmic calcium-dependent entry of extracellular calcium,^108,109 which Dong et al.¹¹⁰ have shown to be caused by the opening of store-operated, CRAC channels. In airway epithelium, activation of the P2Y₂ receptor increases Cl⁻ secretion by opening a calcium-activated chloride channel and the cystic fibrosis transmembrane conductance regulator (CFTR), which has potential relevance to the treatment of cystic fibrosis, a disease that is caused by mutations in the CFTR gene.^111–113 The synthetic P2Y₂ receptor agonist INS37217 has been employed to promote Cl⁻ and water secretion in tracheal epithelium, to increase ciliary beat frequency and mucin release in human airway epithelium¹¹⁴ and to stimulate subretinal fluid reabsorption in a rabbit model of retinal detachment.¹¹⁵ Both the P2Y₂ and P2Y₄ receptors are present in the luminal membrane of mouse distal colonic mucosa and stimulation of these receptors increases K⁺ secretion.²³

In the cochlea, activation of the P2Y₄ receptor in epithelial cells of Reissner's membrane inhibits Na⁺ transport out of endolymph by closing the epithelial Na⁺ channel (ENaC), which the authors speculate might be caused by a decrease in the open probability of ENaC due to depletion of PI(4,5)P₂ in the plasma membrane after P2Y₄ receptor activation.¹¹⁶ The P2Y₄ receptor also controls the lumenal K⁺ concentration in the cochlea by activating apical potassium channels on strial marginal epithelial cells¹¹⁷, whereas P2Y4 inhibits K⁺ currents in cerebral artery myocytes.⁵⁵ P2Y_1,2,6 receptors couple to neuronal N-type Ca²⁺ channels and to M-type K⁺ channels, whereas the P2Y₄ receptor couples more effectively to M-type K⁺ channels than to Ca²⁺ channels and involves the βγ subunits of G_i/o proteins.¹¹⁸

G_i-coupled receptors, including the P2Y_12,13,14 receptors, have been shown to inhibit nociception in peripheral neurons by blocking voltage-gated Ca²⁺ channels.¹¹⁹ Many P2Y receptors, including the P2Y_1,2,4,6,12 subtypes, have been shown to modulate G protein-gated, inwardly rectifying K⁺ (GIRK) channels,¹⁰³ which are known to mediate the antinociceptive actions of opioids in the spine.¹²⁰ In general, opening of GIRK channels by P2Y receptors and other GPCRs is mediated by the βγ subunit of G_i/o proteins, whereas closing of GIRK channels is mediated by the α subunit of G_q proteins.^103,121 The P2Y₁ receptor, which couples primarily to G_q proteins, has been shown to both open and close GIRK channels when expressed and activated in rat sympathetic neurons or CHO cells.^121,122 Furthermore, closing of GIRK channels mediated by the P2Y₁ receptor in these studies was PTX-sensitive, indicating that the P2Y₁ receptor in this instance is capable of coupling to G_i/o proteins. Another potassium channel implicated in pain perception, the two-pore K⁺ channel (K_2P), is generally closed by GPCRs coupled to G_q or G_s and opened by GPCRs coupled to G_i/o.¹²³ Shrestha et al., however, demonstrated that activation of both the G_q-coupled P2Y₁ receptor and the G_i/o-coupled P2Y₁₂ receptor inhibits K_2P activity in tsA201 embryonic kidney cells, which the authors suggest is due to the formation of a P2Y₁–P2Y₁₂ receptor complex that suppresses Gi/_o signaling,⁷⁰ similar to the alteration in G protein signaling observed for the A₁-P2Y₂ receptor complex.⁶⁸

P2Y Receptor Coupling to Receptor and Non-Receptor Tyrosine Kinases

Many studies show that P2Y receptors modulate the activity of growth factor receptors or receptor tyrosine kinases as well as non-receptor tyrosine kinases, such as Src.¹⁰ For example, studies with the P2Y₂ receptor indicate that SH3-binding domains (PXXP) in the C-terminal tail are necessary for the constitutive association with the actin-binding protein, filamin A,⁶⁷ as well as with the transient association and activation of Src.¹²⁴ This, then, is necessary for Src-dependent activation of the focal adhesion-related kinase (RAFTK or Pyk2) and several receptor tyrosine kinases, including the platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR), by the P2Y₂ receptor (see Figure 2).¹²⁴ In endothelial cells, the P2Y₂ receptor uses this Src-dependent mechanism to transactivate the vascular endothelial growth factor receptor-2 (VEGFR-2), leading to increased expression of VCAM-1, a vascular cell adhesion molecule that allows firm attachment of circulating leukocytes to the vascular wall.¹²⁵ Recently, the P2Y₁ receptor was shown to transactivate VEGFR-2 to stimulate endothelial cell tubulogenesis,³⁰ indicating that multipe P2Y receptor subtypes can use VEGFR-2 signaling pathways to affect endothelial cell function.

Figure 2.

Model of P2Y₂ receptor signaling to G_q proteins, ion channels, and growth factor receptors. Stimulation of the P2Y₂ receptor with ATP or UTP causes Gα_q-dependent activation of phospholipase C-β (PLCβ) resulting in the hydrolysis of PtdIns(4,5)P₂ to produce the second messengers Ins(1,4,5)P₃ (IP₃), which mobilizes intracellular Ca²⁺, and diacylglycerol (DAG), which activates protein kinase C (PKC).¹⁰ Downstream signaling molecules include calmodulin (CaM), calmodulin-dependent kinase (CaMK), nitric oxide synthase (NOS), nitric oxide (NO), guanylyl cyclase (GC), cyclic GMP (cGMP), protein kinase G (PKG), cytosolic phospholipase A2 (cPLA₂), arachidonic acid (AA), and prostaglandin E2 (PGE2). The P2Y₂ receptor transactivates several growth factor receptors, including EGFRs, PDGFR, and VEGFR-2, through Src-dependent activation of the focal adhesion-related tyrosine kinase (Pyk2), which phosphorylates growth factor receptors and leads to activation of serine/threonine kinases (e.g., ERK1/2, JNK, p38, LIMK1, and p90RSK) and transcription factors (e.g., CREB, NF-kb, ELK-1, c-FOS, and c-JUN) that regulate genes involved in inflammation, apoptosis, cell differentiation, and so on.¹⁰ Src, which binds to Src homology 3 (SH3) domains in the P2Y₂ receptor,¹²⁴ also is involved in the activation of the monomeric G protein (Rac1) and p21-activated serine kinase (PAK) as well as phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt). The P2Y₂ receptor also transactivates EGFRs by activating metalloproteases (ADAM10 and ADAM17), causing the cleavage and release of EGFR ligands, neuregulin and heparin-binding EGF (HB-EGF), which directly activate EGFRs.^126,127

ATP is released upon epithelial injury and the P2Y₂ receptor is known to be involved in epithelial wound repair by activating EGFRs.^31,126,127 Studies have shown that the P2Y₂ receptor in epithelium activates the metalloproteases ADAM10 and ADAM17, which catalyze the release of the growth factors heparin-binding EGF¹²⁷ and neuregulin.¹²⁶ These growth factors can then activate EGFRs in wounded epithelium to promote tissue repair.¹²⁷

Van Kolan et al.¹²⁸ demonstrated that activation of PKB by the P2Y₁₂ receptor in C6 glioma cells requires the tyrosine kinase activities of the insulin growth factor-I (IGF-I) receptor, Src and Pyk2. Furthermore, in cells transfected with the Gβγ -scavenger β-ARK1/GRK2 or Rap1GAPII, stimulation of the P2Y₁₂ receptor with 2-meSADP failed to enhance PKB phosphorylation demonstrating the requirement of Gβγ-subunits and Rap1 for P2Y₁₂ receptor-mediated PKB activation. In CHO cells, activation of the P2Y₁₂ receptor causes an increase in cell proliferation via pathways involving the activation of PI3K, ERK1/2, and the PDGF-β receptor.⁶²

Conclusion

As described in this review, P2Y receptors for extracellular nucleotides have been shown to regulate cellular functions through the formation of multi-protein signaling complexes with other GPCRs, ion channels, integrins, growth factor receptors, cytoskeletal and junctional proteins, as well as with heterotrimeric and monomeric G proteins and other cytoplasmic signaling molecules. These interactions not only influence intracellular signaling induced by extracellular nucleotides, but also enable an individual P2Y receptor subtype to respond differently to nucleotides, depending on the repertoire of proteins expressed in a particular cell type. Since mammalian cells express 39 distinct G-protein subunits, i.e., 21 α-subunits, 6 β-subunits, and 12 γ-subunits, a myriad of heterotrimeric combinations likely exist that potentially regulate the functional consequences of P2Y receptor activation and the divergence of P2Y receptor-mediated responses between different tissue types. Similarly, tissue-specific expression of P2Y receptor subtypes adds to the complexity of signals transduced by nucleotides in different tissues. Thus, the signaling complexes in which P2Y receptors play a functional role can mediate a wide variety of physiological and pathological effects through protein:protein interactions that are now being revealed, many of which are discussed and diagrammed in this review.