Title: Differential Regulation of the Uridine Nucleotide-activated P2Y4 and P2Y6 Receptors
Abstract: Agonist-promoted regulation of the uridine nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor (P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human astrocytoma cells with the cognate agonist UTP resulted in rapid desensitization of the inositol phosphate response and a 50% loss of cell surface receptors. In contrast, incubation of P2Y6-R-expressing cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the medium of UTP-pretreated cells resulted in rapid and complete recovery of surface P2Y4-R even after 12 h of agonist treatment. Although extended incubation with UDP also caused a loss of surface P2Y6-R, rapid recovery of surface P2Y6-R did not occur following removal of agonist. Pharmacological studies indicated that neither protein kinase C nor other Ca2+-activated kinases were involved in agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R. Mutational analyses were carried out to identify domains involved in agonist-dependent regulation of P2Y4-R. Sequential truncation of the carboxyl-terminal domain revealed that sequence between amino acids 332 and 343 was necessary for UTP-promoted desensitization and internalization. Further mutational analyses of the three serines in this domain confirmed that Ser-333 and Ser-334 play a major role in these agonist-promoted changes in P2Y4-R. Experiments were carried out with [32P]Pi-labeled cells to ascertain the role of phosphorylation in regulation of P2Y4-R. Incubation with UTP for 2 min caused a marked increase in phosphorylation of both the wild-type P2Y4-R and the P2Y4–343 truncation mutant. In contrast, no UTP-promoted phosphorylation of the P2Y4–332 truncation mutant was observed. Taken together, these results demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl terminus of P2Y4-R are important for UTP-dependent phosphorylation, desensitization, and loss of surface receptors. Agonist-promoted regulation of the uridine nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor (P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human astrocytoma cells with the cognate agonist UTP resulted in rapid desensitization of the inositol phosphate response and a 50% loss of cell surface receptors. In contrast, incubation of P2Y6-R-expressing cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the medium of UTP-pretreated cells resulted in rapid and complete recovery of surface P2Y4-R even after 12 h of agonist treatment. Although extended incubation with UDP also caused a loss of surface P2Y6-R, rapid recovery of surface P2Y6-R did not occur following removal of agonist. Pharmacological studies indicated that neither protein kinase C nor other Ca2+-activated kinases were involved in agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R. Mutational analyses were carried out to identify domains involved in agonist-dependent regulation of P2Y4-R. Sequential truncation of the carboxyl-terminal domain revealed that sequence between amino acids 332 and 343 was necessary for UTP-promoted desensitization and internalization. Further mutational analyses of the three serines in this domain confirmed that Ser-333 and Ser-334 play a major role in these agonist-promoted changes in P2Y4-R. Experiments were carried out with [32P]Pi-labeled cells to ascertain the role of phosphorylation in regulation of P2Y4-R. Incubation with UTP for 2 min caused a marked increase in phosphorylation of both the wild-type P2Y4-R and the P2Y4–343 truncation mutant. In contrast, no UTP-promoted phosphorylation of the P2Y4–332 truncation mutant was observed. Taken together, these results demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl terminus of P2Y4-R are important for UTP-dependent phosphorylation, desensitization, and loss of surface receptors. Adenine nucleotides function as neurotransmitters in the peripheral and central nervous systems and as autocrine or paracrine signaling molecules in most other tissues (1Burnstock G. Pharmacol. Rev. 1972; 24: 509-581PubMed Google Scholar, 2Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 3Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (500) Google Scholar). Two large classes of receptors, consisting of the ligand-gated P2X receptors and the G-protein-coupled P2Y receptors (4Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar, 5Fredholm B.B. Abbracchio M.P. Burnstock G. Dubyak G.R. Harden T.K. Jacobson K.A. Schwabe U. Williams M. Trends Pharmacol. Sci. 1997; 18: 79-82Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 6Harden T.K. Barnard E.A. Boeynaems J.M. Burnstock G. Dubyak G.R. Hourani S.M.O. Insel P.A. Girdlestone D. The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media, London1998: 209-217Google Scholar), are broadly distributed and promote a myriad of physiological responses to ATP and ADP, including neurotransmission, muscle contraction, immunological responses, and platelet aggregation (2Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 7Harden T.K. Boyer J.L. Nicholas R.A. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 541-579Crossref PubMed Google Scholar). A complex family of ecto-enzymes rapidly hydrolyze or interconvert extracellular nucleotides thereby either terminating their signaling action or producing an active metabolite of altered P2 receptor selectivity (8Zimmermann H. Drug Dev. Res. 1996; 39: 337-352Crossref Scopus (150) Google Scholar).The existence of extracellular pyrimidinergic signaling, first suggested over a decade ago (9von Kügelgen I. Häussinger D. Starke K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1987; 336: 556-560Crossref PubMed Scopus (100) Google Scholar, 10Seifert R. Schultz G. Trends Pharmacol. Sci. 1989; 10: 365-369Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 11Dubyak G.R. Am. J. Respir. Cell Mol. 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Chem. 1997; 272: 24348-24354Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 18Lazarowski E.R. Harden T.K. Br. J. Pharmacol. 1999; 127: 1272-1278Crossref PubMed Scopus (129) Google Scholar) and by cloning of uridine nucleotide-activated G-protein-coupled receptors (19Lustig K.D. Shiau A.K. Brake A.J. Julius D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5113-5117Crossref PubMed Scopus (529) Google Scholar, 20Communi D. Pirotton S. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1995; 270: 30849-30852Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Nguyen T. Erb L. Weisman G.A. Marchese A. Heng H.H.Q. Garrad R.C. George S.R. Turner J.T. O'Dowd B.F. J. Biol. Chem. 1995; 270: 30845-30848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 22Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The P2Y2-R, which is equipotently activated by ATP and UTP, was the first molecularly identified uridine nucleotide-activated receptor (19Lustig K.D. Shiau A.K. Brake A.J. Julius D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5113-5117Crossref PubMed Scopus (529) Google Scholar). However, the demonstration (23Lazarowski E.R. Harden T.K. J. Biol. Chem. 1994; 269: 11830-11836Abstract Full Text PDF PubMed Google Scholar), and then cloning (20Communi D. Pirotton S. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1995; 270: 30849-30852Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Nguyen T. Erb L. Weisman G.A. Marchese A. Heng H.H.Q. Garrad R.C. George S.R. Turner J.T. O'Dowd B.F. J. Biol. Chem. 1995; 270: 30845-30848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 22Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar) of uridine nucleotide-specific members of the P2Y receptor family placed the pyrimidinergic signaling hypothesis on firm ground. The human P2Y4-R1 is specifically activated by UTP (20Communi D. Pirotton S. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1995; 270: 30849-30852Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Nguyen T. Erb L. Weisman G.A. Marchese A. Heng H.H.Q. Garrad R.C. George S.R. Turner J.T. O'Dowd B.F. J. Biol. Chem. 1995; 270: 30845-30848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 24Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Mol. Pharmacol. 1996; 50: 224-229PubMed Google Scholar) and competitively antagonized by ATP (25Kennedy C. Qi A.-D. Herold C.L. Harden T.K. Nicholas R.A. Mol. Pharmacol. 2000; 57 (, 2000): 929-931Google Scholar); nucleoside diphosphates are inactive. In contrast, the human P2Y6-R is specifically activated by UDP and is insensitive to UTP or other triphosphates (22Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 24Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Mol. Pharmacol. 1996; 50: 224-229PubMed Google Scholar).The biological significance of P2Y receptors that exhibit strict specificity for either UTP or UDP is not yet understood, and evidence for physiological release of UDP in concentrations that activate P2Y6-R is not available. Thus, the extracellular breakdown of UTP to UDP may provide an important source of activating agonist for P2Y6-R (26Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). If metabolic formation of UDP is important, then temporal differences might be expected in both the rapidity by which these receptors are activated upon release of nucleotide and in the length of their activation. The relative capacity of P2Y4-R and P2Y6-R to undergo agonist-induced desensitization also would interplay with possible differences in their rapidity and longevity of activation due to differences in the extracellular presence of their cognate agonists.Little is known about the regulation of P2Y-R in general and of the uridine nucleotide activated P2Y-R in particular. Moreover, P2Y4-R and P2Y6-R differ markedly in potential sites for phosphorylation by second messenger-regulated kinases and GRKs. Thus, we initiated a comparative examination of the agonist-promoted changes in the activities of these receptors and of the mechanisms that underlie these changes. Major differences in the mechanisms of regulation of P2Y4-R versusP2Y6-R are described below. We also report identification of two adjacent serines in the carboxyl terminus of the P2Y4-R receptor that play a major role in agonist-dependent phosphorylation, desensitization, and internalization of this receptor.DISCUSSIONThe observation of a broad range of uridine nucleotide-activated physiological responses (2Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar), the cloning of two receptors that are specifically activated by uridine nucleotides (20Communi D. Pirotton S. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1995; 270: 30849-30852Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Nguyen T. Erb L. Weisman G.A. Marchese A. Heng H.H.Q. Garrad R.C. George S.R. Turner J.T. O'Dowd B.F. J. Biol. Chem. 1995; 270: 30845-30848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 22Chang K. Hanaoka K. Kumada M. Takuwa Y. J. Biol. Chem. 1995; 270: 26152-26158Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), and the demonstration of regulated release of cellular UTP (17Lazarowski E.R. Homolya L. Boucher R.C. Harden T.K. J. Biol. Chem. 1997; 272: 24348-24354Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 18Lazarowski E.R. Harden T.K. Br. J. Pharmacol. 1999; 127: 1272-1278Crossref PubMed Scopus (129) Google Scholar) establish pyrimidinergic signaling as a physiologically important regulatory pathway. The UTP versus UDP specificity of P2Y4-Rversus P2Y6-R adds potential complexity to pyrimidinergic signaling, and we have demonstrated here that these two P2Y-R exhibit very different regulatory properties. The P2Y4-R undergoes rapid agonist-induced desensitization but does not down-regulate. In contrast, the P2Y6-R does not undergo rapid agonist-induced desensitization but does down-regulate. Delineation of the molecular basis of the very different modes of regulation of these two uridine nucleotide-activated receptors will be important to establish. In the current study we have shown that agonist-dependent phosphorylation of either of two adjacent serines in the carboxyl terminus of P2Y4-R contributes an initiating step in both desensitization and internalization of this receptor.The rapidly occurring desensitization of P2Y4-R is a predictable regulatory response of a GPCR and confirms results previously reported by Robaye et al. (30Robaye B. Boeynaems J.M. Communi D. Eur. J. Pharmacol. 1997; 329: 231-236Crossref PubMed Scopus (88) Google Scholar) for P2Y4-R. Our kinetic analyses were of insufficient detail to resolve the time course of occurrence of desensitization from that of loss of surface receptors, but desensitization occurred at least as rapidly as did the receptor trafficking response. Agonist-induced desensitization of phospholipase C-linked GPCR has been exceptionally difficult to quantitate (35Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. Trends Pharmacol. Sci. 1993; 14: 279-285Abstract Full Text PDF PubMed Scopus (108) Google Scholar), because activity cannot be assessed in membranes prepared from agonist-preincubated cells as is the case with studies of adenylyl cyclase-linked receptors. Measurement of intact cell Ins(1,4,5)P3 levels (35Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. Trends Pharmacol. Sci. 1993; 14: 279-285Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 36Nakahata N. Harden T.K. Biochem. J. 1987; 241: 337-344Crossref PubMed Scopus (72) Google Scholar, 37Menniti F.S. Takemura H. Oliver K.G. Putney J.W. Mol. Pharmacol. 1991; 40: 727-733PubMed Google Scholar, 38Willars G.B. McArdle C.A. Nahorski S.R. Biochem. J. 1998; 333: 301-308Crossref PubMed Scopus (37) Google Scholar, 39Willars G.B. Nahorski S.R. Challiss R.A. J. Biol. Chem. 1998; 273: 5037-5046Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) may have certain advantages over quantitation of total inositol phosphates in the presence of LiCl as in the current study. Desensitization of the Ins(1,4,5)P3 response almost certainly occurs faster than that revealed here in a measurement that includes not only Ins(1,4,5)P3 but also all of its downstream metabolites. Thus, we only can conclude that agonist-induced desensitization of P2Y4-R is very rapid and likely occurs prior to loss of surface receptors. That agonist-stimulated accumulation of total inositol phosphates is essentially linear during activation of P2Y6-R expressed in the same cells, provides validation of the qualitative, if not quantitative, aspects of our analyses.The P2Y4/343 carboxyl-terminal truncation mutant retained all of the phenotypical responses of the wild type receptor. In contrast, although UTP activated the P2Y4/332 mutant with properties similar to the wild type receptor, this truncated receptor essentially lost its capacity to undergo UTP-induced desensitization or loss of surface receptors. Although other residues in the 332 to 343 domain could provide these regulatory properties, the serines at positions 333, 334, and 339 are potential targets for kinase-promoted phosphorylation. Our studies showed for the first time that agonist-dependent phosphorylation occurs in a P2Y-R (Fig. 12). Moreover, the occurrence of UTP-stimulated phosphorylation in the P2Y4/343 truncation mutant but not in the P2Y4/332 truncation mutant confirms that the domain between residues 332 and 343 provides the principle sites of agonist-dependent phosphorylation. Mutational analyses of individual or combinations of serines in this domain indicate that Ser-333 and Ser-334 are the important sites of regulation and strongly suggest that phosphorylation of either of these two serines is a key step in agonist-dependent desensitization and loss of surface P2Y4-R. However, our results do not rule out an additional contributing role of phosphorylation at residues elsewhere in P2Y4-R.Although the P2Y4-R exhibits a commonly described phenotype of rapid desensitization and loss of surface receptors, it is unusual in that agonist-promoted down-regulation of this receptor apparently does not occur. That is, following agonist removal from the medium (after times of preincubation up to 12 h) surface receptors were replenished within 30–60 min of incubation. Such results could be a vagary of a cell line engineered to express a recombinant receptor. However, P2Y6-R and P2Y2-R (40Sromek S.M. Harden T.K. Mol. Pharmacol. 1998; 54: 485-494Crossref PubMed Scopus (55) Google Scholar) expressed under the same conditions in 1321N1 cells (and apparently to similar levels as P2Y4-R) both down-regulate. We also have extensively studied down-regulation of the endogenous β2-adrenergic receptor (41Doss R.C. Perkins J.P. Harden T.K. J. Biol. Chem. 1981; 256: 12281-12286Abstract Full Text PDF PubMed Google Scholar, 42Waldo G.L. Doss R.C. Perkins J.P. Harden T.K. Mol. Pharmacol. 1984; 26: 424-429PubMed Google Scholar) and M3 muscarinic receptor (43Harden T.K. Petch L.A. Traynelis S.F. Waldo G.L. J. Biol. Chem. 1985; 260: 13000-13006Abstract Full Text PDF Google Scholar, 44Harden T.K. Heng M.M. Brown J.H. Mol. Pharmacol. 1986; 30: 200-206PubMed Google Scholar) in this same cell line. Thus, the P2Y4-R is a GPCR that both rapidly desensitizes and rapidly recovers from desensitization irrespective of the length of time of activation. It will be important to determine both the physiological significance and structural basis for this unusual property of P2Y4-R.The P2Y6-R falls into a relatively small group of GPCR that do not undergo rapid agonist-induced desensitization. This observation is not entirely surprising, because the P2Y6-R lacks serines and threonines in its third cytoplasmic loop and only a single threonine occurs in the carboxyl-terminal domain. Two serines and two threonines are present in the first cytoplasmic loop, but this domain has not been predictably important in GPCR coupling to heterotrimeric G-proteins or in their agonist-dependent regulation. Simple replacement of the carboxyl-terminal domain of P2Y6-R with that of P2Y4-R did not confer to P2Y6-R a capacity to undergo rapid agonist-induced loss of surface receptors. It is uncertain whether such results reflect a regulation-resistant contribution of noncarboxyl-terminal sequence of P2Y6-R, or whether they simply reflect lack of proper structural context in which the phosphorylation of Ser-333 and Ser-334 apparently produce signals for desensitization/internalization in the P2Y4-R protein.The overriding view of agonist-induced regulatory changes in GPCR signaling has followed from studies of the adenylyl cyclase-coupled β2-adrenergic receptor (45Benovic J.L. Bouvier M. Caron M.G. Lefkowitz R.J. Annu. Rev. Cell Biol. 1988; 4: 405-428Crossref PubMed Scopus (326) Google Scholar, 46Lefkowitz R.J. Cell. 1993; 74: 409-412Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The intuitive model of regulation is one of second messenger-regulated kinases immediately downstream of GPCR feeding back to regulate the activities of cohort proteins of the pathway. This model has been substantiated in studies of the β2-adrenergic receptor by demonstrating that cyclic AMP-dependent protein kinase is in part responsible for agonist-induced desensitization (47Clark R.B. Kunkel M.W. Friedman J. Goka T.J. Johnson J.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1442-1446Crossref PubMed Scopus (105) Google Scholar, 48Clark R.B. Friedman J. Dixon R.A.F. Strader C.D. Mol. Pharmacol. 1989; 36: 343-348PubMed Google Scholar, 49Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar). However, the dominant concept is that members of the GRK family of kinases catalyze receptor phosphorylation to provide the major initiating event in agonist-dependent receptor desensitization and internalization (28Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 29Krupnick J.G. Benovic J.L. Annu. Rev. 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Experiments utilizing pharmacological inhibition or down-regulation of PKC clearly confirmed a role for this second messenger-regulated kinase in agonist-promoted desensitization of inositol lipid signaling (31Fisher S.K. Eur. J. Pharmacol. 1995; 288: 231-250Crossref PubMed Scopus (81) Google Scholar, 52Hepler J.R. Earp H.S. Harden T.K. J. Biol. Chem. 1988; 263: 7610-7619Abstract Full Text PDF PubMed Google Scholar). However, very few of these studies established that the effects of PKC were at the level of the involved GPCR, and PKC could act at multiple levels in the inositol lipid signaling pathway. For example, phospholipase Cβ is phosphorylated by PKC, and its activity is inhibited (53Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 54Filtz T.M. Cunningham M.L. Stanig K.J. Paterson A. Harden T.K. Biochem. J. 1999; 338: 257-264Crossref PubMed Scopus (35) Google Scholar). Nonetheless, several phospholipase C-linked GPCR are phosphorylated in response to activation of protein kinase C (32Raymond J.R. J. Biol. Chem. 1991; 266: 14747-14753Abstract Full Text PDF PubMed Google Scholar, 33Diviani D. Lattion A.L. Cotecchia S. J. Biol. Chem. 1997; 272: 28712-28719Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34Spurney R.F. J. Biol. Chem. 1998; 273: 28496-28503Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).The presence of a potential consensus site (S243) for PKC phosphorylation in the carboxyl-terminal portion of the third cytoplasmic loop of P2Y4-R suggested that PKC might play a role in the marked agonist-induced desensitization and loss of cell surface receptors that occurs upon activation of this receptor. However, incubation of P2Y4-R expressing cells with PMA did not mimic these UTP-promoted effects, and neither down-regulation of PKC nor pharmacological inhibition of its activity altered the time course or extent of agonist promoted loss of surface P2Y4-R. Moreover, serine to alanine mutation of S243 did not modify agonist-dependent regulatory effects. Thus, although UTP induces profound changes in P2Y4-R, little or none of these effects follow from a feed-back involvement from its most proximally activated protein kinase. Because ionomycin also failed to induce changes in UTP responsiveness or modify those occurring during incubation with UTP, Ca2+/calmodulin-regulated kinases also are not involved. GRKs recently have been implicated in regulation of Gq/phospholipase C-coupled GPCR (29Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 55Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar, 56Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 57Kwatra M.M. Schwinn D.A. Schreurs J. Blank J.L. Kim C.M. Benovic J.L. Krause J.E. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 9161-9164Abstract Full Text PDF PubMed Google Scholar), and our experiments that exclude PKC in regulation of P2Y4-R indirectly suggest that GRKs also regulate P2Y4-R. However, coexpression of GRK2 with P2Y4-R in Cos-7 cells had no effect on UTP-dependent desensitization or loss of surface receptors whereas in parallel experiments GRK2 expression markedly augmented desensitization and internalization of the P2Y2-R. 2S. Delaney and T. K. Harden, unpublished results. Perhaps another GRK family kinase, e.g. GRK5, is involved in regulation of P2Y4-R. Alternatively, casein kinase 1α catalyzes agonist-dependent phosphorylation and regulation of the Gq/phospholipase C-coupled M3-muscarinic receptor, and potentially other Gq-linked receptors (58Tobin A.B. Totty N.F. Sterlin A.E. Nahorski S.R. J. Biol. Chem. 1997; 272: 20844-20849Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 59Budd D.C. McDonald J.E. Tobin A.B. J. Biol. Chem. 2000; 275: 19667-19675Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).The P2Y-R family is comprised of a group of five Gq/phospholipase C-linked GPCR that exhibit novel selectivity for extracellular adenine and uridine di- and triphosphates. The ATP- and UTP-activated P2Y2-R was shown previously to undergo agonist-induced internalization (40Sromek S.M. Harden T.K. Mol. Pharmacol. 1998; 54: 485-494Crossref PubMed Scopus (55) Google Scholar,60Garrad R.C. Otero M.A. Erb L. Theiss P.M. Clarke L.L. Gonzalez F.A. Turner J.T. Weisman G.A. J. Biol. Chem. 1998; 273: 29437-29444Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and Garrad et al. (60Garrad R.C. Otero M.A. Erb L. Theiss P.M. Clarke L.L. Gonzalez F.A. Turner J.T. Weisman G.A. J. Biol. Chem. 1998; 273: 29437-29444Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) reported that the capacity of UTP to induce P2Y2-R sequestration was reduced by truncation of the carboxyl terminus. The current work reveals that the subfamily of two pyrimidinergic P2Y-R exhibit very different properties of regulation and that each of these receptors exhibits a property of regulation not common among GPCR. Although our work has not established the identity of the kinase involved in regulation of P2Y4-R, it identifies in P2Y-R for the first time sites of phosphorylation important for agonist-dependent regulation of receptor responsiveness and cellular translocation. It will be important to establish whether phosphorylation per se is sufficient to functionally uncouple P2Y4-R from Gq and to establish the role this phosphorylation plays, for example, in interaction with arrestin, in internalization, and in a recycling response that rapidly and completely replenishes surface P2Y4-R even during extended activation of the receptor. Adenine nucleotides function as neurotransmitters in the peripheral and central nervous systems and as autocrine or paracrine signaling molecules in most other tissues (1Burnstock G. Pharmacol. Rev. 1972; 24: 509-581PubMed Google Scholar, 2Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar, 3Burnstock G. Neuropharmacology. 1997; 36: 1127-1139Crossref PubMed Scopus (500) Google Scholar). Two large classes of receptors, consisting of the ligand-gated P2X receptors and the G-protein-coupled P2Y receptors (4Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. 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