Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63–68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620–23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458. Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63–68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620–23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458. A growing number of proteins containing PDZ 1The abbreviations used are: PDZ, postsynaptic density-95, Discs large, zonula occludens-1; MUPP1, multiple PDZ domain protein 1; 5-HT2C R, serotonin2C receptor; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; BOL, 2-bromo-lysergic acid diethylamide; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; WT, wild type.1The abbreviations used are: PDZ, postsynaptic density-95, Discs large, zonula occludens-1; MUPP1, multiple PDZ domain protein 1; 5-HT2C R, serotonin2C receptor; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; BOL, 2-bromo-lysergic acid diethylamide; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; WT, wild type. domains have been shown to play important roles in the organization and/or regulation of signaling events in cells. PDZ domains (or GLGF repeats) were named after three proteins identified over a decade ago: postsynaptic density-95, Drosophila Discs large, and zonula occludens-1 (3Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 4Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (765) Google Scholar, 5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1277) Google Scholar). These three proteins belong to the membrane-associated guanylate kinase (MAGUK) family of proteins. Most MAGUK proteins contain three PDZ domains, an Src homology 2 domain, and a guanylate kinase-like domain, each having different cellular roles. PDZ domains range from 80 to 100 amino acids in length and typically bind to the carboxyl-terminal sequence of target proteins including receptors, channels, and various signaling molecules to regulate subcellular localization, trafficking, recycling, and/or signaling (6Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1037) Google Scholar, 7Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 8Huber A. Eur. J. Neurosci. 2001; 14: 769-776Crossref PubMed Google Scholar, 9Fanning A.S. Anderson J.M. Curr. Top. Microbiol. Immunol. 1998; 228: 209-233PubMed Google Scholar, 10Kornau H.C. Seeburg P.H. Kennedy M.B. Curr. Opin. Neurobiol. 1997; 7: 368-373Crossref PubMed Scopus (312) Google Scholar). MUPP1, a protein containing 13 putative PDZ domains, was isolated in a yeast two-hybrid screening for proteins that bound to the carboxyl-terminal tail of the 5-HT2C R (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar). MUPP1 is expressed in many tissues, whereas the 5-HT2C R is a brain-specific protein (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar, 11Julius D. MacDermott A.B. Axel R. Jessell T.M. Science. 1988; 241: 558-564Crossref PubMed Scopus (536) Google Scholar). The 5-HT2C R has classically been thought to couple to Gq activation; however, additional G protein families have been implicated, leading to the activation of different downstream signaling pathways including phospholipase A2, C, or D, and various cation channels (12Conn P.J. Sanders-Bush E. J. Neurochem. 1986; 47: 1754-1760Crossref PubMed Scopus (59) Google Scholar, 13Mayer S.E. Sanders-Bush E. Mol. Pharmacol. 1994; 45: 991-996PubMed Google Scholar, 14Kaufman M.J. Hartig P.R. Hoffman B.J. J. Neurochem. 1995; 64: 199-205Crossref PubMed Scopus (58) Google Scholar, 15Chang 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, 16Price R.D. Weiner D.M. Chang M.S. Sanders-Bush E. J. Biol. Chem. 2001; 276: 44663-44668Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17McGrew L. Chang M.S. Sanders-Bush E. Mol. Pharmacol. 2002; 62: 1339-1343Crossref PubMed Scopus (60) Google Scholar). Since PDZ-containing proteins can scaffold many signaling molecules together into a signal transduction complex, the interaction between MUPP1 and the 5-HT2C R was further investigated. The 5-HT2C R contains a PDZ binding motif, Ser458-Ser-Val, at its extreme carboxyl terminus, which is critical for interaction with PDZ 10 of MUPP1 (18Becamel C. Figge A. Poliak S. Dumuis A. Peles E. Bockaert J. Lubbert H. Ullmer C. J. Biol. Chem. 2001; 276: 12974-12982Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In an alternate approach to the yeast two-hybrid system, we independently show that PDZ 10 of MUPP1 is the primary site of interaction for the 5-HT2C R. Serotonin stimulation has previously been shown to promote phosphorylation of the two serine residues of the 5-HT2C R PDZ binding motif, Ser458 and Ser459 (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We therefore hypothesize that phosphorylation of the carboxyl-terminal serines of the 5-HT2C R regulates receptor interaction with MUPP1. To test this hypothesis, we investigated whether a modification of Ser458 and/or Ser459 of the 5-HT2C R carboxyl-terminal tail would alter PDZ 10 interaction. Ser458 and/or Ser459 of the receptor tail were mutated to aspartate to mimic phosphorylation (i.e. introduction of a negative charge). Next, cells expressing 5-HT2C Rs were treated with agonist or antagonist to assess the interaction of the 5-HT2C R with MUPP1. The results of these experiments support our hypothesis that phosphorylation is a key regulator of 5-HT2C R interaction with MUPP1. Furthermore, the results indicated that a significant amount of basal phosphorylation of the receptor may also play a yet undetermined role in regulating PDZ-protein interactions. Polyclonal anti-peptide antibodies against amino acids 419–435 (amino acids RHTNERVARKANDPEPG) of the rat 5-HT2C R were generated as described previously (19Backstrom J.R. Sanders-Bush E. J. Neurosci. Methods. 1997; 77: 109-117Crossref PubMed Scopus (22) Google Scholar). Anti-glutathione S-transferase (GST) antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Overlapping regions of MUPP1 containing two or three PDZ domains (Fig. 1), or one PDZ domain (PDZ 9, 10, or 11) were generated by reverse transcription-PCR, sequenced, and subcloned into pGEX-4T1 (Amersham Biosciences) for expression of GST fusion proteins. MUPP1 PDZ 9, 10, or 11 were also subcloned into pGEMEX-1 (Promega), a T7 gene 10 fusion protein vector. MUPP1 PDZ 9–11 was also subcloned into pcDNA3 (Invitrogen). The 5-HT2C R carboxyl-terminal tail (last 60 amino acids) with or without the PDZ binding motif (Ser458-Ser-Val) and a truncation mutant at residue 445 were subcloned into pGEMEX-1. The 5-HT2C R carboxyl-terminal tail with the PDZ binding motif was also subcloned into pGEX-4T1. The 5-HT2C R carboxyl tail Ser458-Ser-Val (WT) was modified to S458A, S458D, S459D, or S458D/S459D by PCR site-directed mutagenesis and subcloned into pGEMEX-1. Escherichia coli was transformed with pGEX-4T1 constructs, induced to overexpress fusion proteins with isopropyl β-d-thiogalactoside, and analyzed. Bacterial lysates were obtained by first adding cold lysis buffer (50 mm Tris pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 1 mm benzamide, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride) to resuspend the pellets. Resuspended pellets were sonicated for 20 s on ice and centrifuged at 15,000 rpm for 30 min at 4 °C. Proteins were resolved on SDS-PAGE to confirm overexpressed GST fusion protein by Coomassie Blue staining and Western blotting using GST antibodies. pGEMEX-1 constructs were used for coupled transcription and translation using the TnT® in vitro translation system (Promega) in the presence of [35S]methionine (PerkinElmer Life Sciences) according to the supplier's protocol to generate 35S-labeled proteins. Ten micrograms of GST fusion proteins or GST were size-fractionated on SDS-PAGE and transferred onto nitrocellulose membrane. Nitrocellulose membranes were blocked with freshly prepared 1% BSA/phosphate-buffered saline for 1 h at room temperature. Solution was then replaced with 35S-labeled fusion proteins in 1% BSA/phosphate-buffered saline buffer and incubated with nitrocellulose membranes for 16 h at 4 °C. Nitrocellulose membranes were rinsed three times for 20 min at room temperature in 1% BSA/phosphate-buffered saline containing 0.2% Triton X-100. Nitrocellulose membranes were air-dried and exposed to x-ray film or a PhosphorImager screen (Amersham Biosciences) to visualize radiolabeled proteins. Western blot analysis using GST antibodies was used to document similar GST protein levels. NIH-3T3 cells stably transfected with the 5-HT2C R (5-HT2C R/3T3) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) until confluent (20Barker E.L. Sanders-Bush E. Mol. Pharmacol. 1993; 44: 725-730PubMed Google Scholar). Cells were washed four times with Hanks' buffered saline solution (with Ca2+/Mg2+) and then serum starved in serum-free Dulbecco's modified Eagle's medium for 16 h. Cells were treated without or with antagonist (1 μm 2-bromolysergic acid diethylamide (BOL)) for 15 min at 37 °C prior to serotonin addition for 30 min at 37 °C. Medium was then removed, 1 ml of Tris buffer (50 mm Tris, pH 7.6, 0.5 mm EDTA, pH 8.0, 5 μm leupeptin, 1 mm phenylmethylsulfonyl fluoride) was added, and cells were scraped from plates and placed in an Eppendorf tube on ice. Membrane extracts were obtained using 300 μl of Tris buffer containing 1% Triton X-100. Membrane protein concentrations were determined by BCA protein assay (Pierce). Equal amounts of protein were added to the affinity columns. Western blot analysis using GST antibodies was used to determine that similar levels of fusion protein were pulled down. Twenty microliters of glutathione-Sepharose beads (Amersham Biosciences) were washed three times with PD buffer (20 mm HEPES, pH 7.6, 100 mm KCl, 10% glycerol, 0.5 mm EDTA, pH 8.0, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1% Nonidet P-40). Ten micrograms of GST fusion proteins or GST were incubated with the washed glutathione beads for 1 h at 4 °C. Five microliters of 35S-labeled fusion proteins or 50 μg of membrane extracts were added to the GST-glutathione beads and incubated for 2–3 h or overnight, respectively, at 4 °C. After incubation, GST-glutathione beads were washed six times (for assays with 35S-labeled fusion proteins) or three times (for assays with membrane extracts) with PD buffer. For pull-downs from 5-HT2C R/3T3 cell lysates, precipitated protein, containing the 5-HT2C R, was treated with peptide:N-glycanase F before SDS-PAGE (see below). Loading dye (6% SDS, 1% β-mercaptoethanol, 20 mm Tris, pH 6.8, 10% glycerol plus a little bromphenol blue) was added to elute proteins. Eluates were separated on SDS-PAGE and transferred onto nitrocellulose membranes. Autoradiography or PhosphorImager screen was used to visualize radiolabeled proteins. Western blot analysis using GST and 5-HT2C R antibodies were used to document GST fusion proteins and 5-HT2C R, respectively. After serum starvation, cells were treated with 100 nm serotonin for 30 min at 37 °C. After treatment, medium was aspirated to remove the serotonin, and cells were washed four times with Hanks' buffered saline solution. Serum-free Dulbecco's modified Eagle's medium was then added, and cells were incubated for 10 or 30 min. Cells were then lysed, and a pull-down assay was performed as mentioned above. This assay was performed as previously described by Backstrom et al. (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, cells grown to confluence were serum-starved and treated with increasing amounts of serotonin for 15 min at 37 °C. This incubation time was previously shown to be the minimal amount of time that would result in maximal receptor phosphorylation. Cells were then lysed, and membrane extracts containing receptors were prepared. Western blot analysis using 5-HT2C R antibodies were used to document changes in 40- and 41-kDa bands, representative of unphosphorylated and phosphorylated 5-HT2C R, respectively. Following pull-downs from membrane extracts, beads were pelleted and washed once with PD buffer containing 0.1% SDS. Fifteen microliters of PD buffer containing 1% SDS was added to the beads, and the mixture was incubated for 15 min at 37 °C. Then 58 μl of PD buffer was added, and after mixing, 15 μl of PD buffer containing 10% Triton X-100 was added. Finally, 2 μl of peptide:N-glycanase F (Glyko or New England Biolabs) was added, and samples were incubated for 2 h at 37 °C. After deglycosylation, 15 μl of 4× loading dye were added, and samples were incubated at room temperature for 20 min prior to SDS-PAGE. Membrane extracts (50 μg) of untreated or serotonin-treated cells were incubated in PD buffer plus 50 units of calf intestinal (alkaline) phosphatase (New England Biolabs) for 2 h at room temperature. After incubation, pull-down assays were carried out as described above. Alkaline Phosphatase Detection—Nitrocellulose membranes were blocked in 1% BSA/Tris blot buffer (25 mm Tris, pH 7.5, 150 mm NaCl, 0.05% Tween 20, 0.05% NaN3)for1hat room temperature. Membranes were then incubated with GST (1:1000 dilution) or 5-HT2C R (3–5 μg/ml) antibodies in 1% BSA/Tris blot buffer for 2 h to overnight at 4 °C. Membranes were washed three times with Tris blot buffer alone for 10 min. Alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (1:1000 dilution; Jackson Immunolaboratories) were incubated with membranes for2hat room temperature. Membranes were washed three times with Tris blot buffer and once with Tris (150 mm pH 9.4) and then developed with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium. Chemilluminescence Detection—Nitrocellulose membranes were blocked in 8% milk/Tween 20 Tris buffer solution (25 mm Tris, pH 7.4, 137 mm NaCl, 0.27 mm KCl, 0.05% Tween 20) overnight at 4 °C. Membranes were then incubated with GST (1:4000 dilution) or 5-HT2C R (0.5 μg/ml) antibodies in 2% milk/Tween 20 Tris buffer solution overnight at 4 °C. Membranes were washed four times for 5 min with Tween 20 Tris buffer solution. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:20,000 dilution; Jackson Immunolaboratories) were incubated with membranes for 45 min at room temperature. Membranes were washed four times for 15 min with Tween 20 Tris buffer solution and developed with the Pierce Supersignal West Dura® kit according to the supplier's protocol. Horseradish peroxidase signal was analyzed by Bio-Rad Flouro-S, and densitometric analysis was performed by QuantityOne (Bio-Rad) software. All bar graph data was analyzed with Graphpad Prism one-way analysis of variance with Tukey's post-test; p < 0.05 is significant, unless otherwise noted in a figure legend. GST alone was the background control for all GST fusion protein experiments, and graph data presented are background-subtracted. Data represent the means ± S.D. from several independent experiments. The 5-HT2CReceptor Selectively Interacts with MUPP1 PDZ 10 —The carboxyl region of MUPP1 containing the last four PDZ domains (PDZ 10 to PDZ 13) was originally identified by yeast two-hybrid screening for 5-HT2C R-interacting proteins (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar). However, it was unclear which PDZ domain interacted with the 5-HT2C R. Therefore, we set out to identify which domain(s) of MUPP1 interacts with the 5-HT2C R. Overlapping PDZ domain regions of MUPP1 were generated as GST fusion proteins (Fig. 1). Purified GST-MUPP1 PDZ domains were used to pull-down in vitro translated 35S-labeled 5-HT2C R carboxyl terminus fusion protein, which consists of the last 60 amino acids harboring a PDZ binding motif, Ser458-Ser-Val. Fig. 2A illustrates that significantly more receptor tail interacted with PDZ 9–11 and PDZ 9–13. A weak interaction of the receptor tail over GST alone was observed with PDZ 12 and 13, suggesting that PDZ 9–11 is the primary 5-HT2C R interacting region. In protein overlay assays, GST-MUPP1 PDZ domain fusion proteins were blotted and probed with 35S-labeled 5-HT2C R carboxyl terminus, and the 5-HT2C R tail specifically interacted with PDZ 9–11 and PDZ 9–13; no other PDZ domains displayed a significant interaction with the receptor tail (results not shown). Thus, both protein overlay and pull-down assays consistently indicate that PDZ 9–11 is responsible for interacting with the 5-HT2C R tail. To further determine the specific site of interaction, GST fusion proteins of the individual PDZ domains, 9, 10, and 11 were made (Fig. 1). Unfortunately, GST-PDZ 10 was unstable when overexpressed in bacteria. Therefore, the ability to pull down the 35S-labeled 5-HT2C R tail by GST-PDZ 9 or 11 was compared with GST-PDZ 9–11. GST fusion proteins of PDZ 9 or 11 alone were not able to bind to the receptor tail as compared with GST-PDZ 9–11, which contains PDZ 10 (data not shown). In a complementary pull-down experiment, the individual MUPP1 PDZ domains 9, 10, and 11 were in vitro translated with [35S]methionine and pulled down by the 5-HT2C R carboxyl-terminal tail expressed as a GST fusion protein (Fig. 2B). The carboxyl tail of the receptor specifically interacted with PDZ 10 and not PDZ domain 9 or 11, further supporting PDZ 10 as the interacting region for the receptor tail. Next, we questioned whether regions upstream of the extreme carboxyl terminus of the 5-HT2C R are able to confer binding to PDZ 10. To address this question, we generated GST fusion proteins of the 5-HT2C R carboxyl-terminal tail missing only the last three residues (Δ PDZ) and the 5-HT2C R carboxyl-terminal tail ending at residue 445 (i.e. missing the last 15 amino acids). In an overlay assay, [35S]PDZ 9–11 was incubated with the different GST-5-HT2C R carboxyl-terminal tail fusion proteins. As illustrated in Fig. 3, PDZ 9–11 binds to WT but not the 5-HT2C R carboxyl-terminal truncation mutants. Mutation of Ser458 in the 5-HT2CReceptor Reveals Altered PDZ 10 Interaction—Studies previously demonstrated that Ser458 and Ser459 at the extreme carboxyl tail of the 5-HT2C R, the same region of the receptor necessary for PDZ 10 binding, are phosphorylated upon ligand activation (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A function for Ser459 phosphorylation in receptor resensitization was proposed; however, the role for Ser458 phosphorylation is unknown. Based upon crystal structures of PDZ domains (21Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (964) Google Scholar, 22Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar, 23Karthikeyan S. Leung T. Birrane G. Webster G. Ladias J.A. J. Mol. Biol. 2001; 308: 963-973Crossref PubMed Scopus (83) Google Scholar, 24Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (290) Google Scholar, 25Tochio H. Hung F. Li M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 295: 225-237Crossref PubMed Scopus (92) Google Scholar) and data compiled on PDZ binding motifs (26Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar), Ser458 of the 5-HT2C R is predicted to be a critical residue for interacting with PDZ 10. We therefore hypothesized that agonist-mediated phosphorylation of Ser458 disrupts the interaction of MUPP1 PDZ domain 10 and its target, the 5-HT2C R. To test this hypothesis, the serine residues in the receptor tail were replaced with aspartic acid to mimic phosphorylation. The last two serine residues of the 5-HT2C R carboxyl tail (Ser458-Ser459) were modified by PCR site-directed mutagenesis to contain S458A, S458D, S459D, or S458D/S459D substitutions. Wild-type and mutated 5-HT2C R tails were labeled with [35S]methionine and incubated with GST-PDZ 9–11. 5-HT2C R tail mutants containing S458A, S458D, and S458D/S459D substitutions displayed a marked loss of interaction to PDZ 9–11 (Fig. 4A). The S459D mutation, however, retained an ability to interact similar to wild-type interaction (Fig. 4B). These results indicate that Ser458 is an important residue in determining the interaction with PDZ 10. Serotonin Treatment Decreases the Ability of the 5-HT2CReceptor to Interact with PDZ 10 —Results from the 5-HT2C R tail mutants raise the possibility of a dynamic regulation of the interaction between the 5-HT2C R and MUPP1. Thus, we investigated whether agonist stimulation of the 5-HT2C R stably expressed in NIH-3T3 cells would also result in a loss of MUPP1 interaction. To determine whether serotonin stimulation had any effect on MUPP1-receptor interaction, cells were incubated with increasing amounts of serotonin, which have been shown to promote receptor phosphorylation (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The ability of the 5-HT2C R to bind to PDZ 10 was assessed by pull-down assays. Fig. 5A shows that cells treated with serotonin led to a dose-dependent decrease in receptor interaction with MUPP1. A 50% reduction in receptor binding to PDZ 10 was observed with a concentration 100 nm serotonin. Moreover, increasing serotonin concentrations caused a dose-dependent increase in phosphorylated receptor with a concurrent decrease in the amount of unphosphorylated receptor as determined by band shift phosphorylation assays (Fig. 5B). To determine whether the loss of PDZ 10 interaction with the receptor was a consequence of agonist binding with 5-HT2C Rs, cells were preincubated in the absence or presence 1 μm of BOL, a 5-HT2C R antagonist, for 15 min prior to the addition of serotonin. BOL antagonized a subsequent serotonin-mediated decrease in receptor pull-down (Fig. 6), thereby demonstrating that the loss of PDZ 10 interaction is a direct consequence of receptor activation. BOL alone had no effect. Alkaline Phosphatase Treatment of the 5-HT2CReceptor Increases PDZ 10 Interaction and Reveals 5-HT2CReceptor Basal Phosphorylation—The reduction of 5-HT2C R binding to MUPP1 may be the direct result of receptor phosphorylation. We therefore investigated whether treatment of lysate containing receptor with alkaline phosphatase would restore MUPP1 interaction. Cells were treated with agonist, and cell lysates were incubated with alkaline phosphatase prior to pull-down assays. As shown in Fig. 7A, alkaline phosphatase treatment resulted in more receptor pull-down in serotonin-stimulated cells. In the absence of serotonin, alkaline phosphatase treatment doubled the amount of receptor binding to PDZ 10 compared with untreated cells. These findings directly support a role for agonist-induced phosphorylation in disrupting 5-HT2C R binding to MUPP1 as well as uncover a potential function for previously reported basal phosphorylation of the receptor. Phosphorylation of the receptor is reversible; therefore, we investigated the activity of endogenous phosphatases against the receptor by washout experiments. Cells were treated with agonist and then washed thoroughly and incubated in serum-free medium for 10 or 30 min before lysis and pull-down assay. Fig. 7B demonstrates a time-dependent increase in 5-HT2C R binding to MUPP1 (Fig. 7B). These results are consistent with previously published data indicating a time-dependent dephosphorylation of the receptor (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). 5-HT2C Rs are implicated in physiological processes such as cerebrospinal fluid production as well as illnesses and disorders including anxiety, migraines, and eating and sleeping disorders (27Roth B.L. Willins D.L. Kristiansen K. Kroeze W.K. Pharmacol. Ther. 1998; 79: 231-257Crossref PubMed Scopus (258) Google Scholar, 28De Vry J. Schreiber R. Neurosci. Biobehav. Rev. 2000; 24: 341-353Crossref PubMed Scopus (153) Google Scholar). Furthermore, the 5-HT2C R is a target for hallucinogenic drugs such as lysergic acid diethylamide (29Burris K.D. Breeding M. Sanders-Bush E. J. Pharmacol. Exp. Ther. 1991; 258: 891-896PubMed Google Scholar, 30Fiorella D. Helsley S. Lorrain D.S. Rabin R.A. Winter J.C. Psychopharmacology. 1995; 121: 364-372Crossref PubMed Scopus (48) Google Scholar). A thorough understanding of intracellular signaling by the 5-HT2C R, including its interaction with PDZ domain containing proteins, may give insight into the cellular mechanisms that underlie these diverse physiological processes. PDZ domain-containing proteins are involved in the localization of potassium channels and glutamate receptors in the synapse as well as localization of numerous other receptors and proteins (31Shieh B.H. Zhu M.Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 32Shieh B.H. Zhu M.Y. Lee J.K. Kelly I.M. Bahiraei F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12682-12687Crossref PubMed Scopus (69) Google Scholar, 33Adamski F.M. Zhu M.Y. Bahiraei F. Shieh B.H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 34Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (551) Google Scholar, 35Scott K. Zuker C.S. Nature. 1998; 395: 805-808Crossref PubMed Scopus (136) Google Scholar, 36Tsunoda S. Zuker C.S. Cell Calcium. 1999; 26: 165-171Crossref PubMed Scopus (82) Google Scholar). PDZ domains typically bind to the carboxyl termini of target proteins that contain a PDZ binding motif. Interestingly, some PDZ binding motifs have been shown to be phosphorylated (37Cohen N.A. Brenman J.E. Snyder S.H. Bredt D.S. Neuron. 1996; 17: 759-767Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 38Leonoudakis D. Mailliard W. Wingerd K. Clegg D. Vandenberg C. J. Cell Sci. 2001; 114: 987-998Crossref PubMed Google Scholar, 39Cao
