Deletion of the Serotonin 5-HT2C Receptor PDZ Recognition Motif Prevents Receptor Phosphorylation and Delays Resensitization of Receptor Responses

Abstract

Phosphorylation-deficient serotonin 5-HT2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of 32P. Treatment of cells stably expressing ΔPDZ or wild-type 5-HT2C receptors with serotonin produced identical maximal responses and EC50 values for eliciting [3H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing ΔPDZ or wild-type 5-HT2C receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were ∼5-fold lower with ΔPDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, responses of ΔPDZ receptors recovered to wild-type 5-HT2Creceptor levels. Receptors with single mutations at Ser458(S458A) or Ser459 (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT2Creceptor responses. Phosphorylation-deficient serotonin 5-HT2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of 32P. Treatment of cells stably expressing ΔPDZ or wild-type 5-HT2C receptors with serotonin produced identical maximal responses and EC50 values for eliciting [3H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing ΔPDZ or wild-type 5-HT2C receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were ∼5-fold lower with ΔPDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, responses of ΔPDZ receptors recovered to wild-type 5-HT2Creceptor levels. Receptors with single mutations at Ser458(S458A) or Ser459 (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT2Creceptor responses. Dulbecco's modified Eagle's medium Tris-buffered saline bovine serum albumin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Serotonin 5-HT2C (formerly 5-HT1C) receptors exists as several isoforms throughout the brain, due to RNA editing and alternative splicing, and function to stimulate phospholipase C through activation of the G protein Gq (1Chang M. Zhang L. Tam J.P. Sanders-Bush E. J. Biol. Chem. 2000; 275: 7021-7029Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Whereas RNA editing generates isoforms in the second intracellular loop that modify receptor signaling (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar, 3Herrick-Davis K. Grinde E. Niswender C.M. J. Neurochem. 1999; 73: 1711-1717Crossref PubMed Scopus (159) Google Scholar, 4Niswender C.M. Copeland S.C. Herrick-Davis K. Emeson R.B. Sanders-Bush E. J. Biol. Chem. 1999; 274: 9472-9478Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), alternative splicing creates truncated nonfunctional receptors (5Canton H. Emeson R.B. Barker E.L. Backstrom J.R. Lu J.T. Chang M.S. Sanders-Bush E. Mol. Pharmacol. 1996; 50: 799-807PubMed Google Scholar, 6Xie E. Zhu L. Zhao L. Chang L.S. Genomics. 1996; 35: 551-561Crossref PubMed Scopus (94) Google Scholar). In addition, agonists promote phosphorylation of nonedited 5-HT2C receptors (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar), raising the possibility that 5-HT2C receptors are also regulated dynamically. Phosphorylation of G protein-coupled receptors regulates signaling through multiple mechanisms including receptor desensitization, which attenuates second messenger responses. In the case of phospholipase C-linked receptors, phosphorylation has been demonstrated to promote desensitization based on observations that receptors lacking the corresponding phosphorylation sites display sustained phosphoinositide hydrolysis responses relative to wild-type receptors (8Alblas J. van Etten I. Khanum A. Moolenaar W.H. J. Biol. Chem. 1995; 270: 8944-8951Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Diviani D. Lattion A.L. Cotecchia S. J. Biol. Chem. 1997; 272: 28712-28719Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 10Lattion A.L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 11Rao R.V. Roettger B.F. Hadac E.M. Miller L.J. Mol. 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Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) by phosphorylation-deficient mutants relative to wild-type receptors, illustrating that mutation of phosphorylation sites involved in desensitization enhances both initial and secondary responses. Although desensitization of 5-HT2C receptor-mediated responses has been observed in assays that examine phosphoinositide hydrolysis (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar, 17Briddon S.J. Leslie R.A. Elliott J.M. Br. J. Pharmacol. 1998; 125: 727-734Crossref PubMed Scopus (26) Google Scholar), release of intracellular calcium (18Akiyoshi J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar), and Ca2+-activated currents (20Boddeke H.W. Hoffman B.J. Palacios J.M. Hoyer D. Naunyn-Schmiedebergs Arch. Pharmacol. 1993; 348: 221-224Crossref PubMed Scopus (17) Google Scholar), it is unknown whether phosphorylation of the 5-HT2C receptor is involved in desensitization. To address this issue, we identified a 5-HT2C receptor domain that is required for receptor phosphorylation and performed functional assays with phosphorylation-deficient receptor mutants. First, we determined that phosphorylation of the 5-HT2C receptor requires the carboxyl-terminal PDZ (PSD-95 discs-largeZO-1) recognition motif, a domain present in nonedited and edited isoforms of the 5-HT2C receptor. Next, we found that phosphorylation-deficient receptors display identical initial responses as the wild-type 5-HT2C receptor in phosphoinositide hydrolysis and calcium release assays. However, phosphorylation-deficient receptors exhibit diminished secondary responses and a delayed recovery relative to wild-type 5-HT2C receptors. Cells stably expressing 5-HT2C receptors with a single serine-to-alanine mutation also display decreased receptor phosphorylation and diminished secondary calcium responses. We therefore propose that 5-HT2C receptor phosphorylation promotes resensitization of 5-HT2C receptor-mediated responses. The production, purification, and characterization of anti-peptide antibodies against rat 5-HT2C receptors have been described (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar, 22Backstrom J.R. Sanders-Bush E. J. Neurosci. Methods. 1997; 77: 109-117Crossref PubMed Scopus (22) Google Scholar). Antibodies against a region of the third intracellular loop (referred to below as anti-2C-IC antibodies) were generated against amino acids 270–288 (NH2-CKKNGGEEENAPNPNPDQK-COOH) of the rat sequence and purified against a shorter peptide (NH2-CKKNGGEEENAPN-COOH). Antibodies against a region of the carboxyl terminus (referred to below as anti-2C-CT antibodies) were generated against amino acids 419–435 of the rat sequence (NH2-RHTNERVARKANDPEPGC-COOH, with cysteine added to the carboxyl terminus of peptide) and purified against the same peptide. Mutant 5-HT2C receptors were generated from wild-type rat receptor cDNA (INI isoform) (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar) using recombinant Pfupolymerase (Promega, Madison, WI), a forward primer (primer 16.3, 5′-TTGGCATTGTATTCTTTGTGTTTCTGA-3′), and a reverse primer containing anXbaI site. For mutants lacking the PDZ recognition motif (ΔPDZ), polymerase chain reaction was performed with primer 16.3 and the reverse primer 5′-TGTCTAGATTTAAATCCTCTCGCTGACCACATTAG-3′. For the S458A and S459A mutants, two rounds of polymerase chain reaction were performed with primer 16.3 and overlapping reverse primers. The first round was performed with primer 16.3 and 5′-TTACACACTGGCAATCCTCTCGCTGACCACATTAGA-3′ (S458A) or 5′-TTACACGGCACTAATCCTCTCGCTGACCACATT-3′ (S459A), and the second round with primer 16.3 and 5′-TGTCTAGATATTACACACTGGCAATCCTCTCGCT-3′ (S458AXba) or 5′-TGTCTAGAATTTACACGGCACTAATCCTCTCGCT-3′ (S459AXba). For mutants lacking the carboxyl terminus except for the immunodominant region of the 2C-CT epitope and the conserved cysteine (Δ375/CT), two rounds of polymerase chain reaction were performed as described in detail elsewhere. 1J. R. Backstrom, manuscript in preparation. Amplified DNA was digested with StuI and XbaI and ligated into the StuI/XbaI sites of the wild-type 5-HT2C receptor cDNA in a Bluescript plasmid (Stratagene, La Jolla, CA). Sequences were confirmed by automated sequencing at the Core Facility of the Center for Molecular Neuroscience at Vanderbilt University. Receptor cDNA was isolated from the Bluescript plasmid with KpnI and XbaI and ligated into the mammalian expression plasmid pCMV2 (a gift of Dr. David Russell). The carboxyl-terminal sequences of wild-type, ΔPDZ, Δ375/CT, S458A, and S459A 5-HT2Creceptors are illustrated in Fig. 1. NIH 3T3 fibroblasts were transiently transfected with wild-type or mutant 5-HT2C receptor cDNA to determine whether the cellular distribution of mutant receptors differed from that of wild-type 5-HT2C receptors. Fibroblasts electroporated with 25 μg of receptor cDNA were seeded in eight-well Lab-Tek II containing Dulbecco's modified Eagle's medium with bovine serum and Cells were at in a containing for The medium was and with saline containing and After min, was and with saline containing After min, the medium was and the were with Tris-buffered saline and containing bovine serum albumin a second of were at for to antibodies against the 5-HT2C receptor or at a of in containing were added to and for at After with at a of in containing was added to and for at After with and two with were and with were with a in the Cell Core at Vanderbilt University. were from of NIH 3T3 fibroblasts with rat wild-type (INI isoform) (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar) or mutant 5-HT2C receptor Mutant receptor cDNA was with plasmid and cells were with were in containing bovine and were identified by Mutant with receptor to the wild-type 5-HT2C receptor were in subsequent functional assays. Mutant ΔPDZ receptors demonstrated identical for the agonists serotonin and acid as wild-type 5-HT2C receptors in with not Cells in the of serum were with and with of Molecular was added to a of of medium (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). After agonist was and was for min at Cell were and with of containing and The cells were and were in a and at for 10 min at The were and the were with containing 10 and were after as described were determined with the protein assay using as the protein were and with anti-2C-IC or anti-2C-CT antibodies as described protein were with containing and and on were from to in a modified and were with anti-2C-IC or anti-2C-CT and the were using secondary antibodies as described (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar). were on a using the domain NIH at the of Cells stably expressing wild-type or mutant ΔPDZ 5-HT2C receptors were with as described (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar), except cells were with of for Cells were or with for of cells were in and were with of containing and For of receptors, μg of was added to protein and at with of by antibodies to protein in to a of of of was and was for at with The were and with containing the and and and of containing was added to the and the was for min at of containing was added to the and after of and of recombinant Molecular were After for at of was added to the and of was on two was to and were with anti-2C-IC and the was and to a Cells stably expressing ΔPDZ or wild-type 5-HT2C receptors were in medium in the of as described E.L. Westphal R.S. D. Sanders-Bush E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Cells were with of serotonin in the of 10 and 10 for were isolated by and by and EC50 values were with imaging was performed with cells in Cells in containing bovine serum were to The medium was with and Cells were with the calcium Molecular for at in cells were with and using a to a calcium imaging The imaging of a to calcium was by at of and The to the initial with 100 nm serotonin was for 10 Cells were with for or 10 min a second application of 100 nm The for decay of the initial responses were as the maximal and responses. The second of a was by the first to determine the initial for were from from were the agonist Fibroblasts stably expressing the wild-type 5-HT2C receptor were in medium containing to 5-HT2C receptors. Cells were or with serotonin or with serotonin in the of the were with and on were with 5-HT2C receptor antibodies against a region of the third intracellular loop or carboxyl terminus cells protein with a of Treatment of cells with serotonin caused of protein second The serotonin-mediated in from to was by with the third The of protein was on serotonin and was maximal after treatment with 100 were also performed on receptors that were with to Although this also demonstrated in receptor treatment of cells with produced the and treatment of with The possibility that the protein was a of receptors was or was added to from cells with serotonin of decreased the of the protein to were performed to determine the of serotonin-mediated receptor phosphorylation and To examine cells were with serotonin for of the protein was by 10 min of agonist the second Treatment with agonist for third not the of To examine cells were with serotonin for min and After the cells were for in the absence of serotonin of The of protein decreased with a of 10 min the of protein These results are with the of protein phosphorylation and and establish that the protein is a of 5-HT2C of 5-HT2C receptor phosphorylation and the of 5-HT2C receptor phosphorylation after treatment of cells with 100 nm serotonin for 10 min was to that after are of cells with 100 nm serotonin for min were with medium and for the After agonist the of 5-HT2C receptor phosphorylation was 10 min The of are To determine which region of the 5-HT2C receptor was from cells were in the band-shift phosphorylation assay using anti-peptide antibodies against the third intracellular loop and the carboxyl terminus The results were with phosphorylation the carboxyl terminus not two mutant 5-HT2C receptor were that lack two or phosphorylation sites in the carboxyl terminus The receptor was by of amino acids and with a region of the 2C-CT epitope and the conserved cysteine from the To determine whether the cellular distribution of mutants from that of wild-type fibroblasts were transiently transfected with receptor and receptors were with anti-2C-IC Receptors that lack the PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) were throughout transiently transfected cells in a to that of wild-type receptors In receptors that lack of the acid were in the results were observed receptors were with anti-2C-CT antibodies not the intracellular distribution of receptors it to results from phosphorylation ΔPDZ receptor were Fibroblasts stably expressing ΔPDZ or wild-type 5-HT2C receptors were or with serotonin and in two phosphorylation assays. Phosphorylation was not in the band-shift assay using two ΔPDZ and and wild-type receptors were as determined by the in to To the lack of phosphorylation of the ΔPDZ incorporation were performed with receptors in the absence of Cells with were with or and receptors were with anti-2C-IC The were with and to a to determine incorporation of or and with anti-2C-IC antibodies to determine the of receptors not with the results from the band-shift incorporation of was not in ΔPDZ receptors from and the wild-type receptor was To the that phosphorylation of the 5-HT2C receptor promotes desensitization, a phosphoinositide hydrolysis assay was performed with expressing ΔPDZ receptors or wild-type 5-HT2C receptors receptors demonstrated in with identical EC50 values and wild-type, of the PDZ recognition motif not the EC50 or maximal a single application of serotonin relative to the wild-type 5-HT2C receptor. To determine whether treatment of cells with serotonin promotes desensitization of a subsequent we a that was demonstrated to promote desensitization of wild-type 5-HT2Creceptor responses (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). Cells were or with 100 nm a that maximal receptor phosphorylation for After with phosphoinositide hydrolysis was determined with of serotonin to that of cells with serotonin caused a in the EC50 of wild-type receptors and in the EC50 of ΔPDZ receptors. In cells were with serotonin for maximal 5-HT2C receptor phosphorylation occurs 10 min of serotonin treatment calcium imaging was to examine 5-HT2C receptor responses in a the EC50 of serotonin for ΔPDZ and wild-type 5-HT2C values and with wild-type and ΔPDZ, values with stably expressing ΔPDZ or wild-type 5-HT2Creceptors were with in the absence or of 100 nm serotonin for were with medium and with to serotonin in for were isolated by and to The from were by of by values and with wild-type and ΔPDZ, values with in a Cells stably expressing ΔPDZ or wild-type 5-HT2Creceptors were with in the absence or of 100 nm serotonin for were with medium and with to serotonin in for were isolated by and to The from were by of by imaging was performed with cells expressing ΔPDZ or wild-type 5-HT2Creceptors to examine the of receptor desensitization. Cells were with 100 nm serotonin for 10 min, for 2.5 min, and with 100 nm 5-HT2C receptors and ΔPDZ receptors demonstrated a release of intracellular Furthermore, wild-type and ΔPDZ receptors maximal responses and for decay of the initial responses and ΔPDZ, by After for 2.5 min, a second application of serotonin to cells expressing wild-type receptors calcium release at of the initial and In a second application of serotonin to cells expressing ΔPDZ receptors produced responses that were of the initial and To this washout were that after for min, secondary responses of wild-type and ΔPDZ receptors were and of the initial After for 10 min, secondary responses of wild-type and ΔPDZ receptors were receptors display delayed recovery of calcium responses relative to wild-type Cells stably expressing wild-type or ΔPDZ 5-HT2C receptors were in medium and with for Cells were with 100 nm serotonin Fig. at the on the on the was generated by the of the second calcium by the first Cells expressing ΔPDZ receptors demonstrated functional resensitization of calcium responses at 2.5 and for are of at ΔPDZ receptors lack two phosphorylation sites serine-to-alanine mutations were and expressing S458A or S459A receptors were in phosphorylation and calcium release assays. For the phosphorylation cells were with and the of receptors were determined in the band-shift assay S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wild-type receptor results were with 100 nm serotonin not For the calcium release cells were with 100 nm serotonin for 10 min, for min, and with 100 nm Whereas the secondary responses of S458A receptors were to of wild-type receptors, of S459A receptors were diminished mutation of Ser459 decreased receptor phosphorylation and diminished subsequent calcium responses relative to wild-type 5-HT2C receptors. Treatment of cells expressing serotonin 5-HT2Creceptors with serotonin promotes phosphoinositide hydrolysis Sanders-Bush E. Hoffman B.J. S. A. PubMed Scopus Google Scholar), release of intracellular calcium (18Akiyoshi J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar, H. Br. J. Pharmacol. 1999; PubMed Scopus Google Scholar), and receptor phosphorylation (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). Although subsequent responses to agonist application are relative to the first (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar, 17Briddon S.J. Leslie R.A. Elliott J.M. Br. J. Pharmacol. 1998; 125: 727-734Crossref PubMed Scopus (26) Google Scholar, J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar), it is not whether phosphorylation of the 5-HT2C receptor a in desensitization. we 5-HT2C receptor phosphorylation-deficient mutants and serotonin responses in phosphoinositide hydrolysis and calcium release we that of the amino which a PDZ recognition motif (Ser458-Ser-Val-COOH; serotonin-mediated phosphorylation the recovery of 5-HT2C receptor responses. 5-HT2C receptors have of and after treatment of cells with to receptors are with of and (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar). two are from cells in medium containing serum (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar), we the possibility that present in the of receptors. we that the protein is a of 5-HT2C receptors. Cells in medium 5-HT2C receptors with a of treatment of cells with the agonist serotonin the of the agonist is by with a receptor and by agonist washout or treatment of with These results establish that the protein 5-HT2C receptors. We the band-shift phosphorylation assay to a domain of 5-HT2C receptor that is required for receptor truncated 5-HT2C receptors were generated that lack phosphorylation sites in the carboxyl terminus or the two The mutant receptor was by after the domain to amino acids of the carboxyl terminus and with a region of the 2C-CT epitope that and The cysteine from the domain was conserved in this this has been to for the function of receptors H. D. Res. PubMed Scopus Google Scholar). The mutant ΔPDZ receptor amino acids Mutant 5-HT2C receptor cDNA was transiently in and the distribution of receptors was with that of wild-type 5-HT2C receptors. were receptors were ΔPDZ and wild-type 5-HT2C receptors were in a throughout Cell expressing ΔPDZ receptors were therefore a for the of the PDZ recognition motif in phosphorylation assays. The band-shift phosphorylation assay and incorporation were to examine phosphorylation of two expressing ΔPDZ 5-HT2C receptors. In the absence of wild-type and ΔPDZ receptors of Treatment of cells with serotonin caused the of the in wild-type receptors, in was in ΔPDZ receptors. In of the results with the band-shift serotonin-mediated incorporation of was not in ΔPDZ receptors, wild-type receptors demonstrated These results that the band-shift phosphorylation assay 5-HT2C receptor phosphorylation and establish that the PDZ recognition motif is required for receptor we demonstrated that of cells stably expressing wild-type 5-HT2C receptors with serotonin the EC50 for phosphoinositide hydrolysis the maximal to serotonin (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). To determine whether receptor phosphorylation is involved in the observed desensitization, cells stably expressing ΔPDZ or wild-type 5-HT2C receptors were or with 100 nm serotonin and with of with serotonin a in the EC50 of serotonin for ΔPDZ receptors for wild-type 5-HT2C receptors is not with the that 5-HT2C receptor phosphorylation promotes desensitization and the possibility that phosphorylation desensitization or promote resensitization of 5-HT2C receptor responses. The of with serotonin was in the functional assays in the phosphorylation assays and it is that 5-HT2C receptor sites were serotonin calcium imaging was to examine the of 5-HT2Creceptor desensitization and resensitization a that to receptor ΔPDZ and wild-type 5-HT2C receptors in intracellular calcium with maximal responses. In the of responses of wild-type and ΔPDZ receptors with of These results a that desensitization of 5-HT2C receptor responses occurs in the absence of receptor After cells with recovery responses were After min, wild-type 5-HT2C receptor responses recovered to 50% of the initial ΔPDZ receptor responses recovered to of the initial After 10 min, which to the for of wild-type 5-HT2C receptor ΔPDZ and wild-type 5-HT2C receptors were not the results from phosphoinositide hydrolysis and calcium release suggest that phosphorylation of the 5-HT2C receptor enhances resensitization of 5-HT2C receptor responses desensitization ΔPDZ 5-HT2C receptors lack two phosphorylation single mutants were at the PDZ recognition motif or Ser459 to determine mutation of receptor phosphorylation calcium responses. Cell expressing identical of S458A or S459A 5-HT2Creceptors were first in the band-shift phosphorylation S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wild-type 5-HT2C receptor that both and Next, calcium release responses were responses of S459A receptors to a second application of serotonin were diminished relative to wild-type responses of S458A receptors the wild-type These results are with a of Ser459 the PDZ recognition motif in resensitization of 5-HT2C receptor responses. In the PDZ recognition motif of receptors is involved in of receptors to the and mutations this motif receptor H.W. D. A. M. Nature. 1999; PubMed Scopus Google Scholar). observations are with a of Ser459 in resensitization of 5-HT2C receptor responses and suggest that phosphorylation at this regulates We Westphal and for and Dr. Niswender for and