Modulation of ion channel function by P2Y receptors - Springer Link

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R EVIEW ARTICLE

Modulation of Ion Channel Function by P2Y Receptors So Yeong Lee and Scott M. O’Grady* Department of Physiology and the Molecular Veterinary Biosciences Graduate Program, University of Minnesota, Animal Science/Veterinary Medicine, St. Paul, MN 55108

Abstract P2Y receptors are classified as P2 purinergic receptors that belong to the superfamily of G-protein coupled receptors. They are distinguishable from P1 (adenosine) receptors in that they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species. Eight functional subtypes have been characterized. Nucleotide binding produces activation of specific G-proteins that in turn regulate the function of membrane bound enzymes including phospholipase C and adenylyl cyclase. Certain P2Y receptor subtypes possess a PDZ domain located at the end of the C-terminal region of the receptor. PDZ domains have been established as sites for protein-protein interaction, thus providing a possible mechanism for receptor modulation of membrane protein function independent of G-protein activation. In this review we discuss recent findings that suggest that P2Y receptors can modulate the function of ion channels through multiple proteinprotein interactions at the plasma membrane that do not directly involve G-protein activation. Index Entries: G-protein coupled receptor; sulfonylurea receptor; inwardly rectifying K+ channels; CFTR; PDZ domains.

to the P2 class of purinergic receptors. P2 receptors are divided into two groups: P2X receptors function as ligand-activated ion channels and P2Y receptors belong to the superfamily of Gprotein–coupled receptors (1). This proposal was accepted by the International Union of Pharmacology (IUPHAR) Subcommittee in 1994 (2). A comparison of cloned P2Y receptor sequences with other G-protein–coupled receptors indicates that they are more closely related to somatostatin and angiotensin receptors instead of P1 purinergic receptors that are activated by adenosine and related compounds (3–6). The focus of this review is on the

INTRODUCTION Nucleotide phosphates including adenosine diphosphate (ADP), adenosine triphosphate (ATP), uracil diphosphate (UDP), and uracil triphosphate (UTP) have been well established as extracellular signaling molecules that produce multiple effects in a variety of tissues and cell types. The actions of these nucleotides are mediated by cell surface receptors that belong

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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76 G-protein–independent signaling properties of P2Y receptors and how they regulate the function of membrane-associated proteins. At this time, eight subtypes of P2Y receptors have been cloned and functionally characterized. These include the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 receptor subtypes (6–11). Gaps in the numbering sequence have arisen as a result of inclusion of non-mammalian subtypes (for example chick p2y3 and Xenopus p2y8 receptors) (12,13) or isoforms with low sequence homology to P2Y receptors and no reported functional activity following expression (14–16). Like other G-protein-coupled receptors, P2Y receptors possess seven transmembrane (TM) domains with an extracellular N-terminus and intracellular C-terminus. Molecular modeling in combination with site-directed mutagenesis studies of the P2Y1 receptor has shown that TM domains 3, 5, 6, and 7 are involved in nucleotide phosphate binding to the receptor (17–19). In addition, two distinct disulfide linkages between extracellular loop 2 (EL2) and TM domain 3 and EL3 and the N-terminus of the receptor are critical for receptor activation and cell surface expression. A similar disulfide bond is present between EL3 and the N-terminus of the closely related angiotensin II receptor and is thought to be important in maintaining the circular arrangement of TM domains in this receptor (20,21). Based on structure and phylogeny studies, there are two main branches in the P2Y receptor family: one group includes P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors and the other group is composed of P2Y12, P2Y13, and P2Y14 receptors. P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors possess a YQ/KXXR motif located in TM7 whereas P2Y12, P2Y13, and P2Y14 receptors have the KEXXL motif in this region These differences in sequence within TM7 appear to be responsible for the differences in agonist pharmacology between these groups (22). Five of the seven cloned P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptor) are known to increase phospholipase C (PLC) activity through activation of Gq/11 (23–26). Interestingly, the human (h) P2Y11 recepCell Biochemistry and Biophysics

Lee and O’Grady tor has been shown to activate both adenylyl cyclase as well as PLC (27). ATP stimulation of P2Y11 receptors expressed in Chinese hamster ovary cells and HL-60 promyelocytic leukemia cells produced rapid accumulation of cyclic adenosine monophosphate (cAMP), suggesting that the receptor can activate Gs (27,28). Previous studies in platelets have shown that two distinct P2 receptor subtypes were present and play an important role in platelet aggregation (29,30). Platelet stimulation with ADP produced an increase in intracellular Ca2+ and a change in shape that was mediated by activation of P2Y1 receptors (7,31). ADP also stimulated the previously known P2TAC receptor (now identified as P2Y12 receptor (32)) to produce inhibition of adenylyl cyclase activity as a result of Gi activation (31,33,34). It is worth noting that unlike other cloned P2Y receptors, the P2Y12 receptor has not been shown to increase PLC activity or activate Gq (31,33,34). As recently stated by Nicholas (7), sequence analysis of the P2Y12 receptor indicates significant homology to the UDP-glucose receptor as well as several orphan G-protein-coupled receptors. Similar to P2Y12 receptor, P2Y13 receptors are coupled to Gi and inhibit adenylyl cyclase (11) and P2Y14 receptors are orphan G-protein-coupled receptors that are activated by UDP-glucose and couple to the Gi/o class of G-proteins (8). Thus future expression studies may lead to the identification of new P2 receptor subtypes with distinct G-protein-coupling characteristics. In addition to regulating PLC and adenylyl cyclase activities, P2Y receptors have been shown to affect cell proliferation and differentiation by selective activation of extracellular signal–regulated protein kinases (ERK) (35–45). ERKs are members of a family of serine/threonine protein kinases known as mitogen-activated protein kinases (MAPK) (46,47). Activation of the MAPK signaling pathway following purinergic receptor stimulation results in induction of immediate early response genes including c-fos, c-jun, c-myc, and junB leading to long-term changes in gene expression (48). In astrocytes, the ERK/MAPK cascade is stimulated shortly after ATP binds to the receptor

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P2Y Receptors and Ion Channel Function (35). Evidence indicates that ERK activation occurs independently of PLC stimulation and subsequent IP3 formation and involves activation of PLD and translocation of a Ca2+ independent PKC isoform (PKCδ) to the plasma membrane. Inhibition of ERK activity directly or its phosphorylation by PKCδ inhibits purinergic stimulation of DNA synthesis, thus indicating that ERKs mediate mitogenic signaling by at least some P2Y receptor subtypes (35). It is also demonstrated that activation of ERK1/2 via Ras and PKC/Raf signaling pathway by P2Y receptors is Ca2+ independent in astrocytes (49,50). P2Y2 receptor activation in human promonocytic U937 cells with UTP was shown to induce phenotypic differentiation and inhibition of proliferation. UTP stimulated phosphorylation of MEK1/2 and ERK1/2 by activating phosphatidylinositol 3-kinase and csrc. However, unlike astrocytes, ERK1/2 phosphorylation in U937 cells did not appear to be dependent on PKC activation (51). Thus mitogenic signaling mechanisms activated by purinergic agonists appear to involve distinct downstream pathways leading to stimulation of ERK and subsequent regulation of deoxyribonucleic acid (DNA) transcription. It is also known that P2Y receptors are involved in activation of Rho proteins and Rho kinase in rat arterial smooth muscle (52,53). In vascular myocytes, P2Y receptors are coupled to activation of RhoA and stimulate organization of the actin cytoskeleton (54).

P2Y RECEPTOR REGULATION OF ION CHANNELS INVOLVING G-PROTEIN ACTIVATION P2Y receptors have been shown to mediate membrane-delimited G-protein regulation of ion channels. In rat cerebellar neurons, activation of an outwardly rectifying, pertussis toxininsensitive, GDPβS-sensitive K+ current by 2MeS-ATP > ADP > ATP indicated involvement of a P2Y1-like receptor coupled to channel activation by β,γ G-protein subunits (55). Singlechannel currents evoked by 2MeS-ATP in


77 hippocampal neurons occurred without latency, suggesting that the channel was activated by plasma membrane factors and did not involve intracellular components (56). Studies using 2MeS-ATP and ATP for activation of K+ channels in striatal neurons showed that the channels were regulated by PKC-dependent phosphorylation (57). P2Y1 receptors in dorsal root ganglion neurons have been shown to be involved in generation of action potentials by activation of nonselective cation channels in response to touch (58). Agonist stimulation of P2Y receptors are also known to inhibit M-type K+ channels in rat hippocampal neurons, sympathetic neurons, and adrenal cortical cells (59–61). P2Y receptors also modulate a voltagegated K+ current in brown adipocytes (62). Studies in guinea pig ventricular myocytes have shown that treatment with 0.5 µM 2MeSATP produces activation of a monovalent cation channel with an inwardly rectifying current voltage relationship (63). In addition P2Y receptor stimulation inhibits both N-type and L-type Ca2+ currents in ventricular myocytes and sympathetic neurons (64–67). In epithelial cells, P2Y receptor stimulation with UTP produces activation of apical membrane Cl– channels through mobilization of intracellular calcium (68–71). In addition, UTP inhibits amiloride-sensitive Na+ channels in several epithelial cell types by mechanisms involving either increases in intracellular Ca2+ or activation of PKC (72,73). It has been proposed that UTP and related pyrimidergic analogs may serve as potentially useful therapeutic agents for the treatment of cystic fibrosis because these compounds can restore Cl– secretion and reduce hyperabsorption of Na+ in airway epithelia of patients with cystic fibrosis (CF) patients (74–76).

ROLE OF THE PDZ DOMAIN IN RECEPTOR PROTEIN INTERACTIONS AT THE PLASMA MEMBRANE PDZ domains are well-conserved protein sequences that are capable of mediating pro-

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78 tein-protein interactions (76–78). Recent studies with the β-adrenergic receptor and cystic fibrosis transmembrane conductance regulator (CFTR) have shown that the C-terminus of the receptor interacts with a Na+/H+ exchange regulatory factor (NHERF) through a PDZ (PSD-95/Dlg/Zo-1) domain resulting in inhibition of Na+/H+ exchange (NHE) activity (79–81). NHERF homologous, such as EBP50 (ERM (ezrin-radizin-moesin)-binding phosphoprotein 50) and E3KARP (NHE3 kinase A regulatory protein) also have PDZ domains and regulate NHE similar to NHERF (82,83). The ERM family of proteins is associated with the plasma membrane and plays a role in the assembly and stabilization of cell surface cytoskeletal structures (84). The N-terminal domain of ezrin is known to interact with the membrane proteins whereas the C-terminal domain links to cytoskeletal proteins, such as F-actin. Interestingly, the C-terminal sequence of CFTR interacts with PDZ domains of EBP50 or ECKARP and this interaction is important in stabilizing CFTR at the apical membrane of epithelial cells (85,86). Thus, NHERF homologues are examples of adaptor proteins that can modulate the activity of ion channels and membrane transporters, such as the NHE through PDZ domains. The P2Y1 receptor also has a PDZ domain-binding motif (DTSL) at the end of the C-terminal region that can potentially bind to NHERF and possibly modulate NHE activity or ion channels such as CFTR in a manner similar to the β-adrenergic receptor (79,80).

ION CHANNEL INTERACTIONS THAT DO NOT INVOLVE PDZ DOMAINS PDZ domain-independent protein interactions have been suggested as a mechanism for coupling between CFTR and Ca2+ activated Cl– channels (CaCC) (87). When CFTR was expressed in bovine pulmonary artery endothelial cells, activation of the channel with 3isobutyl-1-metyl-xanthine (IBMX) and forskolin produced an increase in CFTR conductance and inhibited ATP-dependent activation of endoge-


Lee and O’Grady nous CaCC channels. Although CFTR has a PDZ domain at the end of the C-terminal sequence, removal of the PDZ domain did not alter the inhibitory effects of activated CFTR on CaCC. The authors speculated that the C-terminal region of the R domain of CFTR was responsible for modulation of CaCC activity and that the PDZ domain was not directly involved. CFTR is also known to inhibit amiloride-sensitive Na+ channels (ENaC) in human colon and airway epithelial cells (88,89). Therefore, in CF, an inherited disease due to impaired expression and function of CFTR, ENaC mediated Na+ absorption is enhanced in airway epithelial cells (88). Although CFTR and EBP50 interact through PDZ domains, the PDZ domain does not appear to be involved in the interactions between CFTR and ENaC. Boucherot et al. (90) suggested that interactions between the two channels involves the first functional nucleotide-binding domain (NBF1) because mutations in the NBF1 region of CFTR resulted in a decreased ability to inhibit ENaC activity. The PDZ domain was not found to be important because CFTR mutants that lack this binding domain were still able to inhibit ENaC after stimulation with IBMX and forskolin. Thus certain ion channels such as CFTR appear to be able to modulate the function of neighboring ion channels through multiple protein–protein interaction sites.

P2Y RECEPTOR-MEDIATED MODULATION OF ION CHANNEL FUNCTION IN XENOPUS OOCYTES Recently, P2Y receptors have been shown to regulate ion channel function independent of PDZ domain interactions in Xenopus oocytes (91–93,97). Expression of P2Y receptors in oocytes was shown to stimulate a slowly activating inward current that was previously identified as the transient inward (Tin) current. Tin current was originally observed following the injection of mRNA from rat brain, cloned 5-HT1a and 5-HT2c receptors or alpha subunit of Gq (94–96). The molecular identity of the Tin channel Volume 39, 2003

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Fig. 1. Voltage dependence of the Tin current elicited by agonist-activated Gq-coupled receptors expressed in Xenopus oocytes. (A) Representative current traces recorded from oocytes expressing the hB1-bradykinin receptor. Oocytes were held at 0 mV and then stepped from –140 mV to +80 mV in 20-mV increments. (B) Normalized conductance as a function of voltage for hB1-bradykinin (n = 13), hP2Y1 (n = 13), hP2Y11 (n = 15), and the sP2Y (n = 12) receptors. (C), Normalized conductance as a function of voltage for Tin currents elicited by activated hB1-bradykinin (n = 13), hP2Y1 (n = 13), and hB1/Y1 chimeric (n = 6) receptors. Modified from ref. 93. currents following activation by Gq and by P2Y receptor activation showed that certain P2Y receptor subtypes were capable of altering the voltage dependence and inactivation gating of the channel. These effects were shown to be dependent on specific sequences within the Cterminal region of the receptor (Figs. 1 and 2).

THE EFFECT OF P2Y RECEPTORS ON THE VOLTAGE DEPENDENCE OF TIN CHANNEL

is presently unknown but its activation has been shown to require I (a) the alpha subunit of Gq, (b) the presence of Ca2+, and (c) strong hyperpolarization of membrane voltage. Comparison of Tin Cell Biochemistry and Biophysics

Previous studies have shown that expression of the alpha subunit of Gq in oocytes alone was sufficient to make the Tin channel sensitive to activation by strong hyperpolarizing voltage pulses (96). Analysis of the conductance-voltage relationship of the Gq-dependent current has shown that it is steeply voltage dependent between –80 and –40 mV and exhibits complete inactivation within 3 s following membrane hyperpolarization (92,93,97). The current exhibits nearly identical characteristics when it is activated following expression of the rat (r) M1-muscarinic receptor and the human (h) B1bradykinin receptor. Interestingly, expression of certain subtypes of P2Y receptors including the hP2Y1, hP2Y11, and skate (s) P2Y receptors produce a significant hyperpolarizing shift in the conductance-voltage relationship that is

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Fig. 2. Effects of the C-terminal domain on voltage sensitivity of the Tin channel. (A) Location of truncation mutations introduced into the hP2Y1 receptor. The last four amino acids, DTSL, represent a consensus class 1 PDZ-binding motif. (B) Normalized conductance as a function of voltage for Tin currents elicited by activated hP2Y1 (n = 13), hP2Y1342tr (n = 18), hP2Y1349tr (n = 9), and hP2Y1369tr (n = 10) receptors. (C) C-terminal sequence comparisons between P2Y receptors. The underlined and bolded amino acids represent potential sequences involved in protein–protein interactions with the Tin channel. hP2Y1, hP2Y11, and sP2Y receptors modulate voltage dependence, whereas hP2Y2 and rP2Y6 receptors modulate inactivation gating (see Fig. 3 and text). Modified from ref. 93.

unique to the properties of these receptors. The P2Y4 pyrimidergic receptor does not produce this effect and is similar to the responses observed with the rM1-muscarinic and hB1bradykinin receptors (92,93,97).


Voltage clamp experiments with an expressed receptor chimera composed of the hB1-bradykinin receptor and the C-terminal sequence of the hP2Y1 receptor (hB1/Y1 chimera) resulted in a current response that

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P2Y Receptors and Ion Channel Function was nearly identical to that previously observed with the native hP2Y1 and hP2Y11 receptors (Fig. 1C). This result indicated that the C-terminal domain of the receptor was involved in modulating Tin channel function. Subsequent experiments with C-terminal truncated hP2Y1 receptors at various sites upstream from the PDZ domain showed that channel modulation required a unique sequence motif located between residues A342 and N360 in the C-terminus of the receptor (ASRRSEANLQSKSEDMTLN) (Fig. 2). A similar consensus sequence was also identified in the sP2Y and hP2Y11 receptors but was absent in all other P2Y receptor subtypes (Fig. 2C). These experiments clearly showed that the PDZ domain was not involved in modulating the voltagesensitivity of the channel. Moreover, The conserved sequence motif present in the C-terminus appears to be a site for P2Y receptor interaction with the Tin channel itself, or possibly, an adaptor protein that could function is an analogous way to NHERF.

EFFECT OF P2Y RECEPTORS ON INACTIVATION GATING OF THE TIN CHANNEL When the hP2Y2 or rP2Y6 receptors were expressed in oocytes, currents elicited by step hyperpolarization to –140 mV did not exhibit complete inactivation when compared to the hP2Y1 receptor or other expressed Gq-coupled receptors (92,93). When the C-terminal sequence of the hP2Y1 receptor was replaced with the hP2Y2 C-terminus (Y1/Y2 chimera), the rate of inactivation of the Tin channel was similar to that of the wild-type hP2Y2 receptor (Fig. 3). Furthermore, when the C-terminus of the hB1-bradykinin receptor was replaced with the C-terminal sequence of the hP2Y2 receptor (hB1/Y2 chimera), inactivation gating was also similar to that of the native hP2Y2 receptor. In contrast, when the C-terminal sequence of the P2Y2 receptor was replaced with the C-terminus of the P2Y1 receptor (Y2/Y1 chimera), the Y2/Y1 chimera exhibited a faster rate of inactiCell Biochemistry and Biophysics

81 vation compared to native hP2Y2 receptors, but inactivation was still significantly slower than native hP2Y1 receptors or the hB1-bradykinin receptor activated currents. This result suggested that the C-terminus of the hP2Y2 receptor was involved in modulating inactivation gating of the Tin channel. When sequences from hP2Y2 and rP2Y6 receptors were compared, a consensus (QRXG/R) sequence motif was identified in the C-terminal region of these receptors and was not found to be present in other P2Y receptor subtypes (Fig. 2C). Therefore, it was concluded that the (QRXG/R) sequence motif was important in modulating the inactivation gating of Tin channel.

RECEPTOR-MEDIATED REGULATION OF VOLTAGE-GATED CATION CHANNELS Modulation of the Tin channel by P2Y receptors appears to be analogous to the previously characterized modulation of an inwardly rectifying K+ channel (Kir6.0 subfamily) by the sulfonylurea receptor. Direct interaction between Kir6.1/Kir6.2 and the SUR1 sulfonylurea receptor has been established in several studies (98–101). In insulin-secreting cells and cerebral cortex, physical coupling between SUR1/ Kir6.1/6.2 was demonstrated in native cells using 125I-labeled 4-azidosalicyloyl analog of glibenclamide for photoaffinity labeling (99). Clement IV et al. (1997) showed a similar result with 125I-azodoglibenclamide when SUR1 was co-expressed with Kir6.1 or Kir6.2 in a heterologous expression system. Co-purification of Kir6.1/Kir6.2 with SUR1 was also demonstrated using Ni2+-agarose chromatography of His-tagged SUR1, supporting the conclusion that a physical interaction between, Kir6.1/ Kir6.2 and SUR1 exists (100,102). Site-directed deletion experiments indicate that the N-terminal region of Kir6.2 and C-terminal domain of SUR1 are important for coupling. An important property of the Kir6.1/Kir6.2/SUR1 complex is its regulation by ATP. Kir6.1 and Kir6.2 by themselves are not regulated by ATP, thus

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Fig. 3. Inactivation gating of receptor-activated Tin currents. (A) Representative traces of the Tin conductance elicited by activated hP2Y1, rP2Y6, hP2Y2, and hY1/Y2 chimeric receptors. Tin channels were monitored for 5 s in the presence of the appropriate receptor agonist. (B) Representative traces of currents elicited by agonist-activated hB1-bradykinin and hB1/Y1, and hB1/Y2 chimeric receptors following step hyperpolarization to –140 mV. (C) Ifinal/Imax is the current amplitude at the end of the voltage pulse (as indicated by the open arrows in part A and B of the figure) divided by the maximum inward current and represents the degree of Tin channel inactivation gating. This ratio was significantly increased in hP2Y2 (n = 10), rP2Y6 (n = 11), hY1/Y2 (n = 7), hY2/Y1 (n = 7), and hB1/Y2 (n = 7) chimeric receptors. From ref. 93.


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P2Y Receptors and Ion Channel Function when the Kir/SUR complex was disrupted by deletion of 14 amino acids located in N-terminal region of Kir6.2 (Kir6.2∆14), ATP sensitivity of the channel was lost and the channel behaved similarly to wild-type Kir6.2. Highaffinity inhibition of the Kir/SUR complex with tolbutamide was eliminated in cells expressing the deletion mutation (Kir6.2∆14), indicating that the N-terminus of Kir6.2 was involved in coupling to the sulfonylurea receptor (101). When the C-terminal region of the sulfonylurea receptor was deleted (SUR∆C), KATP channel function, including the response to tolbutamide, MgADP, and diazoxide was eliminated, but assembly, trafficking, and highaffinity glibenclamide binding to SUR∆C and Kir6.2 was not altered in HEK293 cells, suggesting that C-terminal region of SUR1 is important in coupling to Kir6.2 (98,101). It is interesting to note that multiple sites of interaction exist between SUR1 and the Kir subunits, just as observed between certain P2Y receptors and the Tin channel. Moreover, the sites of interaction that exist between SUR and Kir were shown to be essential for modulation of Kir function by SUR1. Again, a similar situation exists between P2Y receptors and the Tin channel. Therefore, P2Y receptors may represent another example of an ATP-binding protein that can have specific modulatory influences on the function of ion channels. Recently, it was demonstrated that an interaction exists between voltage-gated Na+ channels (Nav1.9) and receptor tyrosine kinase (TrKB) receptors in neurons within the central nervous system (103). This receptor-Na+ channel interaction results in dramatic regulation of Na+ channel function following binding of brain-derived neurotrophic factor (BDNF) to the receptor and exhibits some similarity to Tin channel activation following nucleotide binding to certain subtypes of P2Y receptors. In contrast to P2Y receptor modulation of Tin channel activity, Nav1.9 channels do not activate without stimulation of TrKB receptors by BDNF. The specific molecular interactions between the Na+ channel and TrKB receptor have not been presently identified, but it has


83 been suggested that the proteins are coupled to each other in the plasma membrane.

CONCLUSION Over the past decade, much of the effort in understanding the signaling properties of Gprotein coupled receptors has focused on the role of G-protein subunits as downstream activators of various membrane bound enzymes, ion channels or membrane transporters. The pioneering studies on β-adrenergic receptor interactions with NHERF proteins through a Cterminal PDZ domain has opened up a new and exciting mode of signaling for G-protein-coupled receptors involving membrane delimited modulation of membrane protein function (79,80). The results of investigations with P2Y receptor modulation of ion channel function in Xenopus oocytes extends these studies and has identified novel and potentially important protein–protein interaction sequences that may be involved in regulation of other membrane proteins. It remains to be seen whether this mode of signaling is important for other types of G-protein-coupled receptors, but identification of new protein–protein interaction domains should be helpful in identifying other receptor subtypes that are capable of this mode of signaling.

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