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

Serotonin 5-HT 2C (formerly 5-HT 1C ) 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 G q (1). Whereas RNA editing generates isoforms in the second intracellular loop that modify receptor signaling (2)(3)(4), alternative splicing creates truncated nonfunctional receptors (5,6). In addition, agonists promote phosphorylation of nonedited 5-HT 2C receptors (7), raising the possibility that 5-HT 2C 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 (8 -13). Furthermore, a second application of agonist produces amplified phosphoinositide hydrolysis (14) and calcium release (13,15,16) 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-HT 2C receptor-mediated responses has been observed in assays that examine phosphoinositide hydrolysis (7,17), release of intracellular calcium (18,19), and Ca 2ϩ -activated currents (20), it is unknown whether phosphorylation of the 5-HT 2C receptor is involved in desensitization. To address this issue, we identified a 5-HT 2C 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-HT 2C receptor requires the carboxyl-terminal PDZ (PSD-95 discs-large ZO-1) recognition motif, a domain present in nonedited and edited isoforms of the 5-HT 2C receptor. Next, we found that phosphorylation-deficient receptors display identical initial responses as the wild-type 5-HT 2C 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-HT 2C receptors. Cells stably expressing 5-HT 2C receptors with a single serine-to-alanine mutation also display decreased receptor phosphorylation and diminished secondary calcium responses. We therefore propose that 5-HT 2C receptor phosphorylation promotes resensitization of 5-HT 2C receptor-mediated responses. 2C Receptor Antibodies-The production, purification, and characterization of anti-peptide antibodies against rat 5-HT 2C receptors have been described (21,22). Antibodies against a region of the third intracellular loop (referred to below as anti-2C-IC antibodies) were generated against amino acids 270 -288 (NH 2 -CKKNGGEEENAPNPN-PDQK-COOH) of the rat sequence and purified against a shorter peptide (NH 2 -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 (NH 2 -RHTNERVARKANDPEPGC-COOH, with cysteine added to the carboxyl terminus of peptide) and purified against the same peptide.

5-HT
Generation were generated from wild-type rat receptor cDNA (INI isoform) (2) using recombinant Pfu polymerase (Promega, Madison, WI), a forward primer (primer 16.3, 5Ј-TTGGCATTGTATTCTTTGTGTTTCTGA-3Ј), and a reverse primer containing an XbaI site. For mutants lacking the PDZ recognition motif (⌬PDZ), polymerase chain reaction was performed with primer 16.3 and the reverse primer 5Ј-TGTCTAGATTTA-AATCCTCTCGCTGACCACATTAG-3Ј. 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 5Ј-TTACACACTGGCAATCCTCTCG-CTGACCACATTAGA-3Ј (S458A) or 5Ј-TTACACGGCACTAATCCTCT-CGCTGACCACATT-3Ј (S459A), and the second round with primer 16.3 and 5Ј-TGTCTAGATATTACACACTGGCAATCCTCTCGCT-3Ј (S458A Xba) or 5Ј-TGTCTAGAATTTACACGGCACTAATCCTCTCGCT-3Ј (S459A Xba). For mutants lacking the carboxyl terminus except for the immunodominant region of the 2C-CT epitope and the conserved cysteine (⌬375/CT), two rounds of polymerase chain reaction were performed as described in detail elsewhere. 1 Amplified DNA was digested with StuI and XbaI and ligated into the StuI/XbaI sites of the wild-type 5-HT 2C 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 pCMV 2 (a gift of Dr. David Russell). The carboxyl-terminal sequences of wild-type, ⌬PDZ, ⌬375/CT, S458A, and S459A 5-HT 2C receptors are illustrated in Fig. 1.
Immunocytochemistry-NIH 3T3 fibroblasts were transiently transfected with wild-type or mutant 5-HT 2C receptor cDNA to determine whether the cellular distribution of mutant receptors differed from that of wild-type 5-HT 2C receptors. Fibroblasts electroporated with 25 g of receptor cDNA were seeded in eight-well Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL) containing Dulbecco's modified Eagle's medium (DMEM 2 ; Life Technologies, Inc.) with 9% bovine serum (Hyclone Laboratories, Logan, UT), 5 units/ml penicillin, and 5 g/ml streptomycin (Life Technologies, Inc.). Cells were incubated at 37°C in a humid chamber containing 5% CO 2 for 2 days. The medium was discarded and replaced with phosphate-buffered saline containing freshly prepared 4% (w/v) paraformaldehyde and 4% (w/v) sucrose. After 15 min, fixative was discarded and replaced with phosphatebuffered saline containing 0.1% (v/v) Triton X-100 (Sigma). After an additional 15 min, the medium was discarded, and the wells were washed once with Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, and 150 mM NaCl) containing 2% bovine serum albumin (BSA; Sigma) before adding a second aliquot of TBS/BSA. Slides were incubated at 4°C for 1-2 days to block nonspecific sites. Primary antibodies against the 5-HT 2C receptor (anti-2C-IC or anti-2C-CT) at a final concentration of 0.5 g/ml in TBS containing 1% BSA were added to wells and incubated for 2 h at room temperature. After three washes with TBS, Cy3-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:1000 in TBS containing 1% BSA was added to wells and incubated for 1 h at room temperature. After three washes with TBS and two washes with water, slides were dried and coverslipped with Aqua PolyMount (Polysciences, Inc., Warrington, PA). Confocal images were captured with a Zeiss LSM410 confocal microscope in the Cell Imaging Core at Vanderbilt University.
Cell Band-shift Phosphorylation Assay-Cells grown in the presence of serum were washed four times with serum-free DMEM and replaced with 5 ml of DMEM. Tunicamycin (Roche Molecular Biochemicals) was added to a final concentration of 2 g/ml of medium (7). After 6 h, agonist was added, and incubation was continued for an additional 15 min at 37°C. Cell supernatants were discarded and replaced with 1 ml of phosphate/EDTA (PE) extraction buffer (50 mM NaH 2 PO 4 /Na 3 PO 4 , pH 7.2, containing 5 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 5 M leupeptin, 5 mM Na 4 P 2 O 7 , and 1 mM Na 3 VO 4 ). The cells were scraped, and cell suspensions were placed in a 1.7-ml tube, sonicated, and then centrifuged at 14,000 ϫ g for 10 min at 4°C. The supernatants were discarded, and the pellets were treated with PE extraction buffer containing 10 mM CHAPS and sonicated. Detergentsoluble proteins were collected after centrifugation as described above. Protein concentrations were determined with the BCA protein assay (Pierce) using BSA as the protein standard. Protein samples (25 g) were electrophoresed, blotted, and probed with anti-2C-IC or anti-2C-CT antibodies as described below.
Immunoblotting-Detergent-soluble protein solutions were diluted with 4ϫ sample buffer (250 mM Tris, pH 6.8, containing 8% SDS, 40% glycerol, 4% 2-mercaptoethanol, and bromphenol blue) and separated on 10% SDS-polyacrylamide gels. Proteins were transferred from gels to nitrocellulose membranes Amersham Pharmacia Biotech) in a modified Towbin transfer buffer (25 mM Tris and 192 mM glycine, pH Ϸ8.6). Membranes were probed with anti-2C-IC or anti-2C-CT antibodies, and the immunoreactive bands were detected using alkaline phosphataseconjugated secondary antibodies (Dako Corp., Carpinteria, CA) as described (21). Band intensities were quantitated on a Macintosh computer using the public domain NIH Image program developed at the United States National Institutes of Health.
Immunoprecipitation of 32 P-Labeled 5-HT 2C Receptors-Cells stably expressing wild-type or mutant ⌬PDZ 5-HT 2C receptors were labeled with [ 32 P]orthophosphate as described (7), except cells were incubated with 0.2 mCi of 32 P/ml for 2 h. Cells were untreated or treated with 1 M 5-hydroxytryptamine for 15 min. Membrane fractions of cells were prepared in PE extraction buffer, and detergent-soluble proteins were extracted with 0.3 ml of PE extraction buffer containing 150 mM NaCl, 1% Triton X-100, and 0.1% SDS. For immunoprecipitation of receptors, 1 g of antibody was added to detergent-soluble protein and incubated overnight at 4°C with rocking. Ten l of goat anti-rabbit antibody beads (generated by conjugating Fc fragment-specific antibodies (Jackson ImmunoResearch Laboratories, Inc.) to protein G-agarose (Pierce) in 20 mM dimethyl pimelimidate to a final concentration of 4 mg of antibodies/ml of gel) was added, and incubation was continued for 4 h at 4°C with rocking. The beads were pelleted and washed sequentially with PE extraction buffer containing the following: 0.5% Triton X-100 and 0.5 M NaCl; 0.25% Triton X-100 and 0.1 M NaCl; 0.1% Triton X-100; and 0.1% SDS (one wash each). Fifteen l of PE extraction buffer containing 1% SDS was added to the beads, and the mixture was incubated for 5 min at room temperature. Then, 15 l of PE extraction buffer containing 5% Nonidet P-40 was added to the beads; and after mixing, 90 l of PE extraction buffer and 2 l (0.4 units) of recombinant N-glycosidase F (Roche Molecular Biochemicals) were added. After incubation for 2 h at 37°C, 40 l of 4ϫ sample buffer was added to the suspension, and 70 l of each fraction was electrophoresed on two 10% gels. One gel was transferred to nitrocellulose, and membranes were probed with anti-2C-IC antibodies; and the other gel was dried and exposed to a PhosphorImager cassette (Molecular Dynamics, Inc.).
Phosphoinositide Hydrolysis Assay-Cells stably expressing ⌬PDZ (clone ⌬PDZ-2) or wild-type 5-HT 2C receptors were grown in serum-free medium in the presence of myo-[ 3 H]inositol as described (23). Cells were stimulated with increasing concentrations of serotonin in the presence of 10 mM lithium chloride and 10 M pargyline for 30 min. [ 3 H]Inositol monophosphates were extracted, isolated by anion-exchange chromatography, and quantitated by liquid scintillation counting. Concentration-response curves and EC 50 values were calculated with GraphPAD Prism software.
Calcium Imaging-Calcium imaging was performed with cells in 35-mm plastic culture dishes. Cells in DMEM containing 9% fetal bovine serum were grown to 50 -70% confluence. The medium was replaced with serum-free DMEM and incubated overnight. Cells were loaded with the calcium indicator fura-2/AM (2 g/ml; Molecular Probes, Inc., Eugene OR) for 1 h at room temperature in HEPES/ calcium buffer. Loaded cells were continuously superfused with HEPES/calcium buffer and visualized using a Nikon inverted microscope attached to a Compix calcium imaging system. The imaging system consists of a CCD-72 camera (Dage-MTI, Inc., Michigan City, IN) attached to an IBM compatible computer executing SIMCA Cimaging software (Compix, Inc.). Intracellular calcium was visualized by fluorescence ratio measurements at wavelengths of 340 and 380 nm. The response to the initial challenge with 100 nM serotonin was monitored for 10 min. Cells were then washed with HEPES/calcium buffer for 2.5, 5, or 10 min before a second application of 100 nM serotonin. The half-lives for decay of the initial responses were calculated as the time between maximal and half-maximal responses. The second peak response of a cell was divided by the first peak response to determine the percent initial response for each cell. Responses were obtained from 20 -80 cells/experiment. Results from three independent experiments (means Ϯ S.D.) were plotted versus the time interval between agonist challenges.

Characterization of 5-HT 2C Receptor Phosphorylation-Fi-
broblasts stably expressing the wild-type 5-HT 2C receptor were grown in serum-free medium containing tunicamycin to generate newly synthesized, unglycosylated 5-HT 2C receptors. Cells were either untreated or treated with serotonin or with serotonin in the presence of the antagonist mianserin. Membrane proteins were solubilized with CHAPS detergent and electrophoresed on SDS-polyacrylamide gels. Immunoblots were probed with 5-HT 2C receptor antibodies against a region of the third intracellular loop (anti-2C-IC) or carboxyl terminus (anti-2C-CT). Untreated cells contained an immunoreactive protein with a mass of 40 kDa ( Fig. 2A, first lane). Treatment of cells with serotonin caused formation of an additional 41-kDa immunoreactive protein ( Fig. 2A, second lane). The serotoninmediated shift in mass from 40 to 41 kDa was blocked by preincubation with the antagonist mianserin ( Fig. 2A, third  lane). The amount of 41-kDa protein was dependent on serotonin concentration (Fig. 2B) and was maximal after treatment with 100 nM serotonin. Experiments were also performed on glycosylated receptors that were treated with N-glycosidase F to remove N-linked sugars before electrophoresis. Although this approach also demonstrated an agonist-mediated increase in receptor mass, treatment of cells with tunicamycin produced better resolution between the 40-and 41-kDa bands than did treatment of cell extracts with N-glycosidase F. The possibility that the 41-kDa immunoreactive protein was a phosphorylated form of 40-kDa receptors was examined. Alkaline phosphatase or buffer was added to CHAPS-soluble extracts prepared from cells pretreated with serotonin (Fig. 2C). Increasing concentrations of alkaline phosphatase progressively decreased the mass of the 41-kDa protein to 40 kDa. Additional experiments were performed to determine the time course of serotonin-mediated receptor phosphorylation and dephosphorylation. To examine phosphorylation, cells were treated with serotonin for increasing times before extracting CHAPS-soluble protein. Formation of the phosphorylated 41-kDa protein was complete by 10 min of agonist treatment, the earliest time point examined (Fig. 3A, second lane). Treatment with agonist for 1 h (Fig. 3A, third lane) did not change the amount of 41-kDa protein. To examine dephosphorylation, cells were treated with serotonin for 15 min and washed four times. After the washes, cells were incubated for increasing times in the absence of serotonin before extraction of CHAPS-soluble protein. The amount of 41-kDa protein decreased with a half-life of 10 Ϯ 1 min (n ϭ 3), whereas the amount of 40-kDa protein increased (Fig. 3B). These results are consistent with the reversible actions of protein phosphorylation and dephosphorylation and establish that the 41-kDa protein is a phosphorylated form of unglycosylated 5-HT 2C receptors.
Immunocytochemistry of Mutant 5-HT 2C Receptors-To determine which region of the 5-HT 2C receptor was phosphorylated, tryptic digests from intact cells were evaluated in the band-shift phosphorylation assay using anti-peptide antibodies against the third intracellular loop (anti-2C-IC) and the carboxyl terminus (anti-2C-CT). The results were consistent with phosphorylation within the carboxyl terminus (data not shown). Thus, two mutant 5-HT 2C receptor constructs were created that lack either two (⌬PDZ) or all (⌬375/CT) potential phosphorylation sites in the carboxyl terminus (Fig. 1). The ⌬375/CT receptor was created by deletion of all 86 amino acids and replacement with a region of the 2C-CT epitope and the conserved cysteine 13 residues from the seventh transmembrane domain. To determine whether the cellular distribution of truncation mutants differs from that of wild-type 5-HT 2C receptors, fibroblasts were transiently transfected with receptor cDNA, and immunoreactive receptors were detected with anti-2C-IC antibodies. Receptors that lack the terminal PDZ recognition motif (Ser 458 -Ser-Val-COOH; ⌬PDZ) were distributed throughout transiently transfected cells (Fig. 4B) in a pattern similar to that of wild-type receptors (Fig. 4A). In contrast, receptors that lack 72 residues of the 86-amino acid tail (⌬375/CT) were localized primarily in punctate structures surrounding the nucleus (Fig. 4C). Identical results were observed when receptors were labeled with anti-2C-CT antibodies (data not shown). Since the intracellular distribution of ⌬375/CT receptors would make it difficult to interpret results from phosphorylation assays, only ⌬PDZ receptor cell lines were evaluated further.
Phosphorylation Assays-Fibroblasts stably expressing ⌬PDZ or wild-type 5-HT 2C receptors were untreated or treated with 1 M serotonin and examined in two phosphorylation assays. Phosphorylation was not detected in the band-shift assay using two independent ⌬PDZ clones (⌬PDZ-2 and ⌬PDZ-19) (Fig. 5A, lanes 1 and 2), whereas wild-type receptors were phosphorylated as determined by the increase in mass to 41 kDa (lane 3). To confirm the lack of phosphorylation of the ⌬PDZ receptor, 32 P incorporation studies were performed with glycosylated receptors in the absence of tunicamycin. Cells labeled with 32 P were treated with 1 M serotonin or vehicle (water), and receptors were immunoprecipitated with anti-2C-IC antibodies. The immunoprecipitates were treated with N-glycosidase F, electrophoresed, and either exposed to a Phos-phorImager cassette to determine incorporation of 32 P (Fig. 5B) or blotted onto nitrocellulose and probed with anti-2C-IC antibodies to determine the total amount of receptors (data not shown). Consistent with the results from the band-shift assay, incorporation of 32 P was not detected in ⌬PDZ receptors from either cell line (Fig. 5B, lanes 1 and 2), whereas the wild-type receptor was phosphorylated (lane 3).
Phosphoinositide Hydrolysis Assays-To test the hypothesis that phosphorylation of the 5-HT 2C receptor promotes desensitization, a phosphoinositide hydrolysis assay was performed with cell lines expressing either ⌬PDZ receptors (⌬PDZ-2) or wild-type 5-HT 2C receptors (Fig. 6). Both receptors demonstrated an ϳ4-fold increase in basal inositol monophosphate accumulation with identical EC 50 values (⌬PDZ, 4.7 Ϯ 1.1 nM; and wild-type, 4.7 Ϯ 1.2 nM; n ϭ six experiments). Thus, dele-tion of the PDZ recognition motif did not alter the EC 50 or maximal response during a single 30-min application of serotonin relative to the wild-type 5-HT 2C receptor. To determine whether treatment of cells with serotonin promotes desensitization of a subsequent response, we utilized a protocol that was previously demonstrated to promote desensitization of wildtype 5-HT 2C receptor responses (7). Cells were either untreated or treated with 100 nM serotonin, a concentration that causes maximal receptor phosphorylation (Fig. 2), for 16 h. After washing four times with serum-free DMEM, phosphoinositide hydrolysis was determined with increasing concentrations of serotonin (10 Ϫ10 to 10 Ϫ5 M) to generate concentration-response curves. Table I illustrates that pretreatment of cells with serotonin caused a 3-fold increase in the EC 50 of wild-type receptors and an 8-fold increase in the EC 50 of ⌬PDZ receptors. In these experiments, cells were treated with serotonin for 16 h; however, maximal 5-HT 2C receptor phosphorylation occurs within 10 min of serotonin treatment (Fig. 3). Therefore, calcium imaging was utilized to examine dynamic 5-HT 2C receptor responses in a more relevant time frame.
Calcium Imaging-Calcium imaging was performed with cells expressing ⌬PDZ (⌬PDZ-2) or wild-type 5-HT 2C receptors to examine the dynamics of receptor desensitization. Cells were treated with 100 nM serotonin for 10 min, washed for 2.5 min, and restimulated with 100 nM serotonin. Wild-type 5-HT 2C receptors (Fig. 7A) and ⌬PDZ receptors (Fig. 7B) demonstrated a robust, transient release of intracellular calcium. Furthermore, wild-type and ⌬PDZ receptors displayed similar maximal responses (Fig. 7) and indistinguishable half-lives for decay of the initial responses (wild-type, 62.4 Ϯ 7.9 s; and ⌬PDZ, 75.2 Ϯ 9.5 s; n ϭ 10; p ϭ 0.31 by unpaired Student's t test). After washing for 2.5 min, a second application of serotonin to cells expressing wild-type receptors promoted calcium release at 37 Ϯ 9% of the initial response (Figs. 7A and 8). In contrast, a second application of serotonin to cells expressing ⌬PDZ receptors produced responses that were only 13 Ϯ 9% of the initial signal (Figs. 7B and 8). To further investigate this observation, longer washout periods were examined. Fig. 8 illustrates that after washing for 5 min, secondary responses of wild-type and ⌬PDZ receptors were 50 Ϯ 21 and 7 Ϯ 10% of the initial response, respectively. After washing for 10 min, secondary responses of wild-type and ⌬PDZ receptors were indistinguishable.
Analysis of S458A and S459A Receptors-Since ⌬PDZ receptors lack two potential phosphorylation sites (Ser 458 -Ser-Val-COOH), serine-to-alanine point mutations were created, and cell lines expressing S458A or S459A receptors were examined in phosphorylation and calcium release assays. For the phosphorylation assay, cells were treated with 1 M serotonin, and the levels of phosphorylated receptors were determined in the band-shift assay (Fig. 9A). Both S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wildtype receptor levels (Fig. 9B, white bars). Identical results were obtained with 100 nM serotonin (data not shown). For the calcium release assay, cells were treated with 100 nM serotonin for 10 min, washed for 5 min, and restimulated with 100 nM serotonin. Whereas the peak secondary responses of S458A receptors were similar to those of wild-type receptors, those of S459A receptors were diminished (Fig. 9B, gray bars). Thus, mutation of Ser 459 decreased receptor phosphorylation and diminished subsequent calcium responses relative to wild-type 5-HT 2C receptors. DISCUSSION Treatment of cells expressing serotonin 5-HT 2C receptors with serotonin promotes phosphoinositide hydrolysis (24), release of intracellular calcium (18,19,25), and receptor phos-  ⌬PDZ and wild-type 5-HT 2C receptors Cells stably expressing ⌬PDZ (⌬PDZ-2) or wild-type 5-HT 2C receptors were labeled overnight with myo-[ 3 H]inositol in the absence (Ϫ) or presence (ϩ) of 100 nM serotonin for 16 h. Wells were washed four times with serum-free medium and stimulated with 10 Ϫ10 to 10 Ϫ5 M serotonin in triplicate wells for 30 min. Radiolabeled inositol monophosphates were isolated by anion-exchange chromatography and subjected to scintillation counting. The data (mean Ϯ S.D.) from four independent experiments were statistically analyzed by two-way analysis of variance, followed by unpaired two-tailed Student's t test. 5 7. Calcium imaging of ⌬PDZ and wild-type 5-HT 2C receptors. Cells stably expressing either wild-type or ⌬PDZ 5-HT 2C receptors were incubated in serum-free medium overnight and then loaded with fura-2 for 1 h. Cells were challenged by a 10-min application of 100 nM serotonin (as illustrated by the bars), washed for 2.5 min, and challenged with serotonin again (arrows). A, calcium responses of cells expressing wild-type receptors showed a robust signal in response to a second serotonin challenge. B, ⌬PDZ receptors did not resensitize to wild-type receptor levels in response to a second challenge with serotonin. Traces are representative of at least three independent experiments. phorylation (7). Although subsequent responses to agonist application are attenuated (desensitized) relative to the first response (7,(17)(18)(19), it is not known whether phosphorylation of the 5-HT 2C receptor plays a role in desensitization. Therefore, we created 5-HT 2C receptor phosphorylation-deficient mutants and examined serotonin responses in phosphoinositide hydrolysis and calcium release experiments. Here, we demonstrate that deletion of the terminal three amino acids, which include a PDZ recognition motif (Ser 458 -Ser-Val-COOH; ⌬PDZ), abrogates serotonin-mediated phosphorylation and, unexpectedly, delays the recovery (resensitization) of desensitized 5-HT 2C receptor responses.
Immunoreactive 5-HT 2C receptors have masses of 51-68 kDa; and after treatment of cells with tunicamycin to prevent N-linked glycosylation, receptors are detected with masses of 40 and 41 kDa (21). Since two bands are detected from cells maintained in medium containing serum (21), we examined the possibility that serotonin, present in serum, alters the migration of receptors. Here, we demonstrate that the 41-kDa protein is a phosphorylated form of 40-kDa 5-HT 2C receptors. Cells in serum-free medium contain 5-HT 2C receptors with a mass of 40 kDa, whereas treatment of cells with the agonist serotonin causes the appearance of the 41-kDa protein. This agonist effect is blocked by co-incubation with mianserin, a 5-HT 2A/2C receptor antagonist, and reversed by either agonist washout or treatment of cell extracts with alkaline phosphatase. These results establish that the 41-kDa protein reflects phosphorylated 5-HT 2C receptors. We exploited the band-shift phosphorylation assay to identify a domain of 5-HT 2C receptor that is required for receptor phosphorylation.
Two truncated 5-HT 2C receptors were generated that lack either all potential phosphorylation sites in the carboxyl terminus (⌬375/CT) or the terminal two serine residues (⌬PDZ). The mutant ⌬375/CT receptor was created by truncation after the seventh transmembrane domain to remove all 86 amino acids of the carboxyl terminus and replacement with a region of the 2C-CT epitope that lacks serine, threonine, and tyrosine residues. The cysteine located 13 residues from the predicted membrane domain was conserved in this construct because this residue has been shown to be important for the function of 5-HT 2A receptors (26). The mutant ⌬PDZ receptor lacks only three amino acids (Ser 458 -Ser-Val-COOH). Mutant 5-HT 2C receptor cDNA was transiently expressed in fibroblasts, and the distribution of immunoreactive receptors was compared with that of wild-type 5-HT 2C receptors. Two different phenotypes were observed: ⌬375/CT receptors were predominantly intracellular, whereas ⌬PDZ and wild-type 5-HT 2C receptors were distributed in a similar pattern throughout cells. Cell lines expressing ⌬PDZ receptors were therefore a valid system for evaluating the contribution of the PDZ recognition motif in phosphorylation assays.
The band-shift phosphorylation assay and incorporation of 32 P were used to examine phosphorylation of two independent cell lines expressing ⌬PDZ 5-HT 2C receptors. In the absence of serotonin, wild-type and ⌬PDZ receptors had an apparent mass of 40 kDa. Treatment of cells with serotonin caused the appearance of the phosphorylated 41-kDa form in wild-type receptors, whereas no increase in mass was detected in ⌬PDZ receptors. In support of the results obtained with the band-shift assay, serotonin-mediated incorporation of 32 P was not detected in ⌬PDZ receptors, whereas wild-type receptors demonstrated robust phosphorylation. These results further confirm that the band-shift phosphorylation assay reflects 5-HT 2C receptor phosphorylation and establish that the PDZ recognition motif is required for receptor phosphorylation.
Previously, we demonstrated that overnight pretreatment of cells stably expressing wild-type 5-HT 2C receptors with serotonin increases the EC 50 value for stimulating phosphoinositide hydrolysis without changing the maximal response to serotonin (7). To determine whether receptor phosphorylation is involved in the observed desensitization, cells stably expressing ⌬PDZ or wild-type 5-HT 2C receptors were either untreated or treated with 100 nM serotonin overnight, washed, and then stimulated with increasing concentrations of serotonin. Interestingly, pretreatment with serotonin promoted a significantly greater increase in the EC 50 value of serotonin for ⌬PDZ receptors (8-fold) than for wild-type 5-HT 2C receptors (3-fold). This result is not consistent with the hypothesis that 5-HT 2C receptor phosphorylation promotes desensitization and raises the intriguing possibility that phosphorylation may actually attenuate desensitization or promote resensitization of 5-HT 2C FIG. 8. ⌬PDZ receptors display delayed recovery of calcium responses relative to wild-type 5-HT 2C receptors. Cells stably expressing either wild-type or ⌬PDZ 5-HT 2C receptors were incubated in serum-free medium overnight and then loaded with fura-2 for 1 h. Cells were challenged twice with 100 nM serotonin (see Fig. 7 ) at the interval indicated on the x axis. A ratio (shown on the y axis) was generated by dividing the peak height of the second calcium response receptor responses. The time of pretreatment with serotonin was much longer in the functional assays (hours) than in the phosphorylation assays (minutes), and it is possible that additional 5-HT 2C receptor sites were phosphorylated during prolonged serotonin treatment. Therefore, calcium imaging was used to examine the dynamics of 5-HT 2C receptor desensitization and resensitization within a time frame that corresponds to receptor phosphorylation. Both ⌬PDZ and wild-type 5-HT 2C receptors displayed robust, transient increases in intracellular calcium with similar maximal responses. In the continued presence of serotonin, responses of wild-type and ⌬PDZ receptors decayed with indistinguishable half-lives of ϳ1 min. These results lend additional support, within a relevant time frame, that desensitization of 5-HT 2C receptor responses occurs in the absence of receptor phosphorylation. After washing cells with agonist-free medium, dramatically different recovery responses were observed. After 5 min, wild-type 5-HT 2C receptor responses recovered to 50% of the initial response, whereas ⌬PDZ receptor responses recovered to only 7% of the initial response. After 10 min, which corresponds to the half-life for reversal of wild-type 5-HT 2C receptor phosphorylation, significant differences between ⌬PDZ and wild-type 5-HT 2C receptors were not observed. Thus, the results from phosphoinositide hydrolysis and calcium release experiments suggest that phosphorylation of the 5-HT 2C receptor enhances resensitization of 5-HT 2C receptor responses rather than altering desensitization kinetics.
Since ⌬PDZ 5-HT 2C receptors lack two potential phosphorylation sites, single point mutants were created at Ser 458 , the PDZ recognition motif serine, or Ser 459 to determine if mutation of either residue alters receptor phosphorylation and/or calcium responses. Cell lines expressing identical densities of S458A or S459A 5-HT 2C receptors were first examined in the band-shift phosphorylation assay. Both S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wild-type 5-HT 2C receptor levels, suggesting that both Ser 458 and Ser 459 are phosphorylated. Next, calcium release responses were examined. Interestingly, responses of S459A receptors to a second application of serotonin were diminished relative to wild-type 5-HT 2C receptors, whereas responses of S458A receptors reproduced the wild-type phenotype. These results are consistent with a major role of Ser 459 rather than the PDZ recognition motif per se in resensitization of 5-HT 2C receptor responses. In contrast, the PDZ recognition motif of ␤ 2 -adrenergic receptors (Ser-Leu-Leu-COOH) is involved in efficient recycling of internalized receptors to the cell surface, and mutations within this motif enhance agonist-mediated receptor degradation (27). Our observations are consistent with a role of Ser 459 in resensitization of 5-HT 2C receptor responses and suggest that phosphorylation at this site regulates resensitization.