Title: Interaction of Calmodulin with the Serotonin 5-Hydroxytryptamine2A Receptor
Abstract: The 5-hydroxytryptamine2A (5-HT2A) receptor is a Gq/11-coupled serotonin receptor that activates phospholipase C and increases diacylglycerol formation. In this report, we demonstrated that calmodulin (CaM) co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts in an agonist-dependent manner and that the receptor contains two putative CaM binding regions. The putative CaM binding regions of the 5-HT2A receptor are localized to the second intracellular loop and carboxyl terminus. In an in vitro binding assay peptides encompassing the putative second intracellular loop (i2) and carboxyl-terminal (ct) CaM binding regions bound CaM in a Ca2+-dependent manner. The i2 peptide bound with apparent higher affinity and shifted the mobility of CaM in a nondenaturing gel shift assay. Fluorescence emission spectral analyses of dansyl-CaM showed apparent KD values of 65 ± 30 nm for the i2 peptide and 168 ± 38 nm for the ct peptide. The ct CaM-binding domain overlaps with a putative protein kinase C (PKC) site, which was readily phosphorylated by PKC in vitro. CaM binding and phosphorylation of the ct peptide were found to be antagonistic, suggesting a putative role for CaM in the regulation of 5-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPγS binding to NIH-3T3 cell membranes, supporting a possible role for CaM in regulating receptor-G protein coupling. These data indicate that the serotonin 5-HT2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function. The 5-hydroxytryptamine2A (5-HT2A) receptor is a Gq/11-coupled serotonin receptor that activates phospholipase C and increases diacylglycerol formation. In this report, we demonstrated that calmodulin (CaM) co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts in an agonist-dependent manner and that the receptor contains two putative CaM binding regions. The putative CaM binding regions of the 5-HT2A receptor are localized to the second intracellular loop and carboxyl terminus. In an in vitro binding assay peptides encompassing the putative second intracellular loop (i2) and carboxyl-terminal (ct) CaM binding regions bound CaM in a Ca2+-dependent manner. The i2 peptide bound with apparent higher affinity and shifted the mobility of CaM in a nondenaturing gel shift assay. Fluorescence emission spectral analyses of dansyl-CaM showed apparent KD values of 65 ± 30 nm for the i2 peptide and 168 ± 38 nm for the ct peptide. The ct CaM-binding domain overlaps with a putative protein kinase C (PKC) site, which was readily phosphorylated by PKC in vitro. CaM binding and phosphorylation of the ct peptide were found to be antagonistic, suggesting a putative role for CaM in the regulation of 5-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPγS binding to NIH-3T3 cell membranes, supporting a possible role for CaM in regulating receptor-G protein coupling. These data indicate that the serotonin 5-HT2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function. The serotonin 5-hydroxytryptamine2A (5-HT2A) 1The abbreviations used are: 5-HT, 5-hydroxytryptamine; R, receptor; CaM, calmodulin; ERK, extracellular signal-regulated protein kinase; GPCR, G protein-coupled receptor; i2, 20-amino acid peptide fragment from the second intracellular loop of the human serotonin 5-HT2A receptor; ct, 20-amino acid peptide fragment from the juxtamembrane region of the carboxyl terminus of the human serotonin 5-HT2A receptor; MEK, mitogen-activated protein kinases kinase; MOPS, 3-(N-morpholino)propanesulfonic acid; PKA, protein kinase A; PKC, protein kinase C; PVDF, polyvinylidene fluoride; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ct-P, a synthetic peptide identical to the ct peptide but containing a phosphorylated threonine at residue 386. receptor is a prototypical G protein-coupled receptor (GPCR) that plays diverse roles in both the central nervous system and peripheral vasculature. In the central nervous system these receptors are widely distributed, being expressed in the neocortex, claustrum, mammilary nuclei, basal ganglia, and anterior cingulate cortex (1Quinn J.C. Johnson-Farley N.N. Yoon J. Cowen D.S. J. Pharmacol. Exp. Ther. 2002; 303: 746-752Crossref PubMed Scopus (57) Google Scholar). 5-HT2A receptors are also highly expressed in vascular smooth muscle and renal mesangial cells, where they mediate contraction and proliferation (2Roth B.L. Nakaki T. Chuang D.M. Costa E. J. Pharmacol. Exp. Ther. 1986; 238: 480-485PubMed Google Scholar, 3Watts S.W. Yeum C.H. Campbell G. Webb R.C. J. Vasc. Res. 1996; 33: 288-298Crossref PubMed Scopus (63) Google Scholar, 4Greene E.L. Houghton O. Collinsworth G. Garnovskaya M.N. Nagai T. Sajjad T. Bheemanathini V. Grewal J.S. Paul R.V. Raymond J.R. Am. J. Physiol. 2000; 278: F650-F658Crossref PubMed Google Scholar), and platelets, where they contribute to aggregation and adherence (5Cook Jr., E.H. Fletcher K.E. Wainwright M. Marks N. Yan S.Y. Leventhal B.L. J. Neurochem. 1994; 63: 465-469Crossref PubMed Scopus (148) Google Scholar), as well as in kidney (6Nebigil C.G. Garnovskaya M.N. Casanas S.J. Mulheron J.G. Parker E.M. Gettys T.W. Raymond J.R. Biochemistry. 1995; 34: 11954-11962Crossref PubMed Scopus (32) Google Scholar) and skeletal muscle (7Guillet-Deniau I. Burnol A.-F. Girard J. J. Biol. Chem. 1997; 272: 14825-14829Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 8Hajduch E. Dombrowski L. Darakhshan F. Rencurel F. Marette A. Hundal H.S. Biochem. Biophys. Res. Commun. 1999; 257: 369-372Crossref PubMed Scopus (25) Google Scholar). The heterogeneous expression of the 5-HT2A receptor is accompanied by a diverse array of pathophysiological implications for 5-HT2A receptor signaling, including roles in sleep, hallucinogenesis, schizophrenia, appetite control, neuroendocrine secretions, hypertension, and depression (9Boess F.G. Martin I.L. Neuropharmacology. 1994; 33: 275-317Crossref PubMed Scopus (599) Google Scholar, 10Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar, 11Zifa E. Fillion G. Pharmacol. Rev. 1992; 44: 401-458PubMed Google Scholar, 12Mann J.J. Arango V. Marzuk P.M. Theccanat S. Reis D.J. Br. J. Psychiatry. 1989; : 7-14Crossref Google Scholar). The 5-HT2A receptor is involved in the mechanism of action of hallucinogens, atypical neuroleptics, antidepressants, and other psychoactive drugs. 5-HT2A receptors signal primarily through heterotrimeric proteins of the Gq/11 subfamily to the activation of phospholipase C, and the subsequent formation of diacylglycerol and activation of protein kinase C (13Conn P.J. Sanders-Bush E. Neuropharmacology. 1984; 23: 993-996Crossref PubMed Scopus (148) Google Scholar, 14Roth B.L. Nakaki T. Chuang D.M. Costa E. Neuropharmacology. 1984; 23: 1223-1225Crossref PubMed Scopus (115) Google Scholar, 15Tamir H. Hsiung S.C. Yu P.Y. Liu K.P. Adlersberg M. Nunez E.A. Gershon M.D. Synapse. 1992; 12: 155-168Crossref PubMed Scopus (31) Google Scholar). Other second messengers and effectors regulated by the 5-HT2A receptor include phospholipase A2 (16Berg K.A. Maayani S. Clarke W.P. Mol. Pharmacol. 1996; 50: 1017-1023PubMed Google Scholar, 17Berg K.A. Maayani S. Goldfarb J. Clarke W.P. Ann. N. Y. Acad. Sci. 1998; 861: 104-110Crossref PubMed Scopus (60) Google Scholar, 18Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), phospholipase D (19Kurscheid-Reich D. Throckmorton D.C. Rasmussen H. Am. J. Physiol. 1995; 268: F997-F1003PubMed Google Scholar), Ca2+ channels (20Eberle-Wang K. Braun B.T. Simansky K.J. Am. J. Physiol. 1994; 266: R284-R291Crossref PubMed Google Scholar, 21Jalonen T.O. Margraf R.R. Wielt D.B. Charniga C.J. Linne M.L. Kimelberg H.K. Brain Res. 1997; 758: 69-82Crossref PubMed Scopus (39) Google Scholar, 22Watts S.W. Ann. N. Y. Acad. Sci. 1998; 861: 162-168Crossref PubMed Scopus (54) Google Scholar), reactive oxygen and nitrogen species (23Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar, 24Matsuda H. Li Y. Yoshikawa M. Life Sci. 2000; 66: 2233-2238Crossref PubMed Scopus (34) Google Scholar), and Na+/H+ exchange (25Saxena R. Saksa B.A. Fields A.P. Ganz M.B. Am. J. Physiol. 1993; 265: F53-F60PubMed Google Scholar, 26Garnovskaya M.N. Nebigil C.G. Arthur J.M. Spurney R.F. Raymond J.R. Mol. Pharmacol. 1995; 48: 230-237PubMed Google Scholar). In essentially all cases, activation of downstream signaling molecules has been shown to be mediated by heterotrimeric G proteins. Calmodulin (CaM) is a small (148 amino acids, ∼17 kDa), soluble protein, which functions as the major calcium sensor in most cells (27Saimi Y. Kung C. Ann. Rev. Physiol. 2002; 64: 289-311Crossref PubMed Scopus (295) Google Scholar). As a prototypical member of the EF-hand family of Ca2+-binding proteins, CaM can bind up to four Ca2+ ions, which subsequently extend the protein to expose hydrophobic patches capable of binding cellular targets (28Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (803) Google Scholar, 29Wriggers W. Mehler E. Pitici F. Weinstein H. Schulten K. Biophys. J. 1998; 74: 1622-1639Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). These targets number well over one hundred and include enzymes, ion channels, transcription factors, and cytoskeletal proteins. Recently, CaM has been shown to bind to several plasma membrane receptors, including the epidermal growth factor receptor, platelet glycoprotein VI, and some GPCRs (30Li H. Villalobo A. Biochem. J. 2002; 362: 499-505Crossref PubMed Scopus (41) Google Scholar, 31Andrews R.K. Suzuki-Inoue K. Shen Y. Tulasne D. Watson S.P. Berndt M.C. Blood. 2002; 99: 4219-4221Crossref PubMed Scopus (78) Google Scholar). The first GPCR that was shown to interact with CaM was the metabotropic glutamate subtype 5 receptor, which contains a CaM-binding site in a region of the extended carboxyl terminus of the receptor also known to bind G protein βγ subunits (32El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Subsequently, the interaction of CaM with the third intracellular loop of D2-dopamine and μ-opioid receptors was shown to regulate receptor coupling to Pertussis toxin-sensitive heterotrimeric G proteins (33Bofill-Cardona E. Kudlacek O. Yang Q. Ahorn H. Freissmuth M. Nanoff C. J. Biol. Chem. 2000; 275: 32672-32680Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 34Wang D. Sadee W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), whereas Nickols et al. (35Nickols H.H. Shah V.N. Chazin W.J. Limbird L.E. J. Biol. Chem. 2004; 279: 46969-46980Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) showed that the interaction of CaM with the V2-vasopressin receptor modulates ligand-induced elevations in intracellular calcium. Our group recently showed that CaM interacts with the Gi/o-coupled 5-HT1A receptor at two distinct sites in the receptor third intracellular loop in regions that overlap with protein kinase C phosphorylation sites (36Turner J.H. Gelasco A.K. Raymond J.R. J. Biol. Chem. 2004; 279: 17027-17037Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Interestingly, binding of CaM to synthetic peptides corresponding to these regions prevented phosphorylation of the peptides by protein kinase C. Furthermore, binding of CaM to the 5-HT1A receptor decreases G protein coupling as assayed by GTPγS binding to crude membrane preparations. 2J. H. Turner and J. R. Raymond, unpublished data. These examples indicate that CaM interactions may play important and diverse roles in GPCR signaling. CaM has previously been shown to be a major target for 5-HT2A receptor signaling. Agonist-mediated up-regulation of the 5-HT2A receptor is dependent upon both CaM and CaM-dependent kinase 2 (37Iken K. Cheng S. Fargin A. Goulet A.C. Kouassi E. Cell. Immunol. 1995; 163: 1-9Crossref PubMed Scopus (126) Google Scholar). Berg et al. (38Berg K.A. Clarke W.P. Chen Y. Ebersole B.J. McKay R.D. Maayani S. Mol. Pharmacol. 1994; 45: 826-836PubMed Google Scholar) showed that CaM is required for 5-HT2A receptor-mediated formation of cAMP in A1A1 cells. Likewise, the CaM-dependent enzymes CaM-dependent kinase 2 and calcineurin (a CaM-dependent phosphatase) play roles in 5-HT-induced cyclooxygenase 2 mRNA expression in renal mesangial cells (39Stroebel M. Goppelt-Struebe M. J. Biol. Chem. 1994; 269: 22952-22957Abstract Full Text PDF PubMed Google Scholar, 40Goppelt-Struebe M. Hahn A. Stroebel M. Reiser C.O. Biochem. J. 1999; 339: 329-334Crossref PubMed Scopus (28) Google Scholar). Finally, the 5-HT2A receptor activates extracellular signal-regulated mitogen-activated protein kinases through the intermediate action of Ca2+/CaM (1Quinn J.C. Johnson-Farley N.N. Yoon J. Cowen D.S. J. Pharmacol. Exp. Ther. 2002; 303: 746-752Crossref PubMed Scopus (57) Google Scholar). Our group previously reported that CaM interacts with the Gi/o-coupled 5-HT1A receptor third intracellular loop at two distinct sites and that the interaction of CaM with those sites may play a role in regulating receptor phosphorylation and desensitization induced by protein kinase C (36Turner J.H. Gelasco A.K. Raymond J.R. J. Biol. Chem. 2004; 279: 17027-17037Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We were interested in establishing whether the Gq/11-coupled 5-HT2A receptor could also interact with CaM, and if so, what consequences this binding might have on receptor function. In the current work, we report that, in NIH-3T3 fibroblasts, CaM co-immunoprecipitates in an agonist-dependent manner with the 5-HT2A receptor. A search of the primary sequence revealed that the 5-HT2A receptor contains two novel CaM-binding motifs, located in the second intracellular loop and the juxtamembrane region of the carboxyl terminus of the receptor. Both motifs contain consensus phosphorylation sites and are important for G protein coupling, indicating that interaction of CaM with those sites could play roles in regulating receptor function. In this report, we sought to characterize the putative CaM-binding sites in the 5-HT2A receptor and to determine their functional significance. Materials—Purified bovine brain calmodulin, biotinylated calmodulin, and purified rat brain PKC were obtained from Calbiochem (La Jolla, CA). Rabbit polyclonal antibody directed against the amino terminus (amino acids 22–41) of the rat 5-HT2A receptor was purchased from Calbiochem. Rabbit polyclonal antibody directed against the carboxyl terminus of the rat 5-HT2A receptor (amino acids 428–443) was kindly provided by Ryan Strachan (Cleveland, OH) and Dr. Bryan Roth (Cleveland, OH). Mouse anti-CaM antibodies were from Upstate Biotechnology (Charlottesville, VA). Dansyl chloride was purchased from Molecular Probes (Eugene, OR) and [35S]GTPγS was purchased from PerkinElmer Life Sciences. Synthesis of 5-HT2A Receptor Peptides—Peptides derived from the amino acid sequence of the second intracellular loop (amino acids 183–202, HSRFNSRTKAFLKIIAVWTI) and carboxyl terminus (amino acids 377–396, PLVYTLFNKTYRSAFSRYIQ) of the human 5-HT2A receptor were synthesized using standard solid-phase methods on a Rainin PS3 automated peptide synthesizer by the Medical University of South Carolina Peptide Synthesis Facility. Peptide sizes and purity were verified using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. When necessary, peptides were purified on a Waters Delta Prep 3000 chromatography system using a C-18 silica column and elution across a linear gradient of acetonitrile in water containing 0.1% (w/v) trifluoroacetic acid (Emory University Microchemical Facility, Atlanta, GA). Cell Culture—NIH-3T3 cells were maintained in minimum essential medium supplemented with 10% fetal calf serum, streptomycin (100 μg/ml), and penicillin (100 units/ml). Cells were incubated at 37 °C in a 5% CO2-enriched, humidified atmosphere. 24–48 h before each experiment, cells were switched to serum-free medium containing 0.5% fatty acid free bovine serum albumin (Sigma). Immunoprecipitation—Quiescent NIH-3T3 cell monolayers grown on 100-mm dishes were treated with agonist (1 μm 5-HT) or vehicle for the appropriate time then lysed in 500 μl of modified radioimmune precipitation assay buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 1 mm NaF, 1 mm Na3VO4, 1 mm phenylmethanesulfonyl fluoride, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin). Lysates were homogenized, clarified by centrifugation at 14,000 × g for 15 min, then pre-cleared by incubation with protein A/G-agarose for 30 min at 4 °C. Pre-cleared lysates were then incubated with commercial anti-5-HT2A receptor antibody overnight at 4 °C. Immunocomplexes were captured by incubation with protein A/G-agarose for 2 h at 4 °C, collected by centrifugation, and washed three times with radioimmune precipitation assay buffer and twice with phosphate-buffered saline. Immunoprecipitates were then resuspended in 2× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE. Gels were analyzed by immunoblot with either anti-CaM antibody or a second anti-5-HT2A receptor antibody. Gel Shift Assays—Gel shift analyses of CaM-peptide complexes were performed using urea-polyacrylamide gel electrophoresis, as described by Erickson-Viitanen and Delgrado (41Erickson-Viitanen S. DeGrado W.F. Methods Enzymol. 1987; 139: 455-478Crossref PubMed Scopus (132) Google Scholar). Reactions (30 μl of total volume) containing 300 pmol of CaM (∼5 μg) and increasing amounts of peptide (0–3000 pmol) were incubated in 100 mm Tris-HCl, pH 7.5, 4 m urea and either 0.1 mm CaCl2 or 1 mm EGTA at 22 °C for 30 min. 15 μl of a 50% glycerol/0.1% bromphenol blue loading buffer was added to each reaction, and the samples were resolved on 14% polyacrylamide gels containing 4 m urea and either 0.1 mm CaCl2 or 1 mm EGTA in the running buffer. Protein was visualized by staining with Gel-code blue (Pierce) staining reagent. Blot Overlay Assays—Peptides (1–100 nmol) were immobilized to PVDF membranes by slot blot and washed twice with 100 mm Tris-HCl, pH 7.5. The membranes were blocked with 5% bovine serum albumin in 100 mm Tris-HCl, pH 7.5, containing 0.1% Tween 20 for 1 h at room temperature, and then were incubated with 0.5 μg/ml biotinylated CaM in the presence of either 0.1 mm CaCl2 or 1 mm EGTA overnight at 4 °C. The PVDF membranes were then washed 3× in the same buffer without CaM, followed by incubation with alkaline phosphatase-conjugated avidin for 1 h at room temperature. Detection was with a chemiluminescent reagent. Fluorometric Measurements with Dansyl-CaM—Dansyl-CaM was synthesized according to the method of Bertrand et al. (42Bertrand B. Wakabayashi S. Ikeda T. Pouyssegur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar). Briefly, 10 mg of CaM was incubated with ∼1 mg of dansyl chloride for 1 h at 4 °C. Dansyl-CaM was purified from unincorporated dye using a Centricon™ concentrator with a molecular mass cutoff of 10,000 Da. Measurement of absorbance at 340 nm (molar extinction coefficient, 3,400 m–1 cm–1) gave an incorporation of ∼1.3 dansyl units per CaM molecule. Fluorescence emission spectra of dansyl-CaM were measured from 400–600 nm using a SLM 8000™ C spectrofluorometer (AMINCO-Bowman) with an excitation wavelength of 340 nm. Test peptides (0–2 μm) were incubated with dansyl-CaM in 100 mm Tris-HCl, pH 7.5, supplemented with 0.1 mm CaCl2 for 2 h at room temperature. The concentration of dansyl-CaM (0.1–0.5 μm) was varied, and concentration-response curves were generated for fluorescence enhancement at each dansyl-CaM concentration. The apparent KD values for each concentration of dansyl-CaM were fit to the Hill equation by linear regression to calculate true affinities. In Vitro Kinase Assays—Thirty-five ng (∼0.06 unit) of purified rat brain PKC was incubated with increasing concentrations of ct peptide (0–6 μm) in kinase buffer (20 mm MOPS, pH 7.2, 25 mm β-glycerolphosphate, 1 mm dithiothreitol, 1 mm CaCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 100 μm [γ-32P]ATP) in a total volume of 25 μl. In some cases, purified bovine brain calmodulin (0–20 μm) was added to reactions. Assays were started by the addition of 5 μCi of [γ-32P]ATP, and then incubated at 30 °C for 1 h. Reactions were then transferred to nitrocellulose squares, washed several times with 0.75% phosphoric acid, followed by a final wash with 100% acetone. Bound radioactivity was measured using liquid scintillation counting. Preparation of NIH-3T3/5-HT2A Receptor Cell Membranes—NIH-3T3 cells overexpressing the human 5-HT2A receptor (8 pmol/mg) were kindly provided by Dr. Elaine Sanders-Bush (Nashville, TN). Cells were grown to confluence in 100-mm dishes, incubated in serum-free medium for 16–24 h, scraped into lysis buffer (100 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1 μg/ml each of aprotinin, leupeptin, and pepstatin), then subjected to homogenization by twenty strokes in a Dounce homogenizer. Lysed cells were centrifuged at 1,000 × g for 10 min to remove whole cells and nuclear debris, and the supernatant was centrifuged at 37,000 × g for 20 min. The resulting pellet was washed twice in lysis buffer and resuspended in 50 mm Tris HCl, pH 7.4, 2.5 mm MgCl2 at a final protein concentration of ∼4 mg/ml. Membranes were frozen by immersion in liquid nitrogen, and stored at –80 °C for future use. [35S]GTPγS Binding to NIH-3T3/5-HT2A Receptor Cell Membranes— The protocol for measurement of [35S]GTPγS binding to crude cell membranes was adapted from the methods of Cussac et al. (43Cussac D. Newman-Tancredi A. Duqueyroix D. Pasteau V. Millan M.J. Mol. Pharmacol. 2002; 62: 578-589Crossref PubMed Scopus (99) Google Scholar) and Adlersberg et al. (44Adlersberg M. Arango V. Hsiung S. Mann J.J. Underwood M.D. Liu K. Kassir S.A. Ruggiero D.A. Tamir H. J. Neurosci. Res. 2000; 61: 674-685Crossref PubMed Scopus (41) Google Scholar). Briefly, 50-μg aliquots of NIH-3T3/5-HT2A receptor cell membranes were added to 500 μl of assay buffer (20 mm HEPES, pH 7.4, 6 mm MgCl2, 50 μm GDP, 100 mm NaCl, 0.5 mm dithiothreitol, 0.1 mm CaCl2), in the presence or absence of increasing concentrations of i2 or ct peptides, CaM, or S-100 protein, and then incubated for 15 min to allow for GDP loading. Reactions were initiated by the addition of [35S]GTPγS (1250 Ci/mmol) to a final concentration of 200 pm, in the presence or absence of 10 μm 5-HT, and incubated for an additional 1 h at 25 °C. Bound and free nucleotides were then separated by filtration over glass fiber filters, and bound radioactivity was determined by scintillation counting. Statistical Analysis—Results shown represent the means ± S.E. of the number of experiments indicated in each case. Statistical analysis was performed by Student's t test. Interaction of CaM with the 5-HT2A Receptor in NIH-3T3 Fibroblasts—Several groups, including ours, have previously shown that CaM plays a role in numerous 5-HT2A receptor signaling pathways, including the activation of mitogen-activated protein kinases (1Quinn J.C. Johnson-Farley N.N. Yoon J. Cowen D.S. J. Pharmacol. Exp. Ther. 2002; 303: 746-752Crossref PubMed Scopus (57) Google Scholar) and Na+/H+ exchange (26Garnovskaya M.N. Nebigil C.G. Arthur J.M. Spurney R.F. Raymond J.R. Mol. Pharmacol. 1995; 48: 230-237PubMed Google Scholar, 45Garnovskaya M.N. Mukhin Y.V. Turner J.H. Vlasova T.M. Ullian M.E. Raymond J.R. Biochemistry. 2003; 42: 7178-7187Crossref PubMed Scopus (25) Google Scholar). As a receptor that couples to Gq/ll type proteins, the 5-HT2A receptor is capable of stimulating phosphoinositide turnover and to subsequently increase intracellular Ca2+ levels. We wondered whether the 5-HT2A receptor might exert some of its Ca2+-sensitive and/or -insensitive intracellular effects by directly interacting with CaM. Other GPCRs, including the D2 dopamine (32El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), μ-opioid receptors (34Wang D. Sadee W. Quillan J.M. J. Biol. Chem. 1999; 274: 22081-22088Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and 5-HT1A receptor (36Turner J.H. Gelasco A.K. Raymond J.R. J. Biol. Chem. 2004; 279: 17027-17037Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), have been shown to directly interact with CaM through their third intracellular loops. We treated wild-type NIH-3T3 cells and NIH-3T3 cells overexpressing the 5-HT2A receptor, with 1 μm serotonin for 5 min, and immunoprecipitated the receptor with a commercial 5-HT2A receptor antibody. As shown in Fig. 1, a CaM-specific mouse monoclonal antibody detected a band at ∼19 kDa in NIH-3T3–5HT2AR cell immunoprecipitates, which corresponds to the Ca2+-bound form of CaM. This immunoreactive band was only moderately detectable in immunoprecipitates from untreated NIH-3T3/5-HT2AR cells, but was significantly increased in immunoprecipitates from cells treated with 5-HT. A second anti-5-HT2AR antibody detected an immunoreactive band at ∼60 kDa in immunoprecipitates from receptor-expressing NIH-3T3 cells that was unchanged after treatment with 5-HT. No immunoreactive bands were detected with either antibody in immunoprecipitates from wild-type NIH-3T3 cells. These data suggest that the 5-HT2A receptor can complex with CaM in an agonist-dependent manner in NIH-3T3 fibroblasts. Identification of Putative CaM Binding Regions in the 5-HT2A Receptor—NMR studies have shown that CaM binds target peptides via hydrophobic interactions and via salt bridges typically involving glutamate residues in the EF-hand regions (47Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (942) Google Scholar, 48Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1179) Google Scholar). Predictably, identified CaM binding regions conform to short peptides that form amphipathic α-helices composed of hydrophobic and positively charged amino acid residues (49O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (717) Google Scholar). Using a web-based computer search program, which identifies such regions based on evaluation criteria such as hydropathy, α-helical propensity, residue charge, helical class, residue weight, and hydrophobic residue content (50Yap K.L. Kim J. Truong K. Sherman M. Yuan T. Ikura M. J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref PubMed Scopus (464) Google Scholar), we identified two putative CaM binding regions in the protein sequence of the 5-HT2A receptor. The illustration in Fig. 2A indicates that the putative CaM-binding domains are localized to the second intracellular loop and the juxtamembrane region of the carboxyl terminus of the receptor. Both regions contain a propensity of hydrophobic and basic amino acids, typical of standard CaM binding regions. Although CaM binding regions do not conform to a linear arrangement of amino acids, several different CaM-binding motifs have been identified based on distances between key hydrophobic residues. The CaM binding region in the second intracellular loop of the 5-HT2A receptor was classified as a 1-8-14 motif, which consists of hydrophobic residues at positions 1, 8, and 14; whereas the putative CaM-binding domain in the carboxyl terminus of the 5-HT2A receptor was classified as a 1-10 motif (Fig. 2B), with key hydrophobic residues separated by eight amino acids. As shown in Fig. 2B, the 5-HT2A receptor CaM binding regions could be aligned with other well defined CaM binding motifs from other proteins. To illustrate the amphipathic nature of the putative CaM-binding sequences of the 5-HT2A receptor, we created helical wheel diagrams using a web-based modeling program (50Yap K.L. Kim J. Truong K. Sherman M. Yuan T. Ikura M. J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref PubMed Scopus (464) Google Scholar). As expected, like most CaM-binding sites, both helical wheels showed clusters of positively charged amino acids on one side of the α-helix, with mostly hydrophobic amino acids concentrated on the opposite side (data not shown). CaM Binds to Peptides Derived from the Second Intracellular Loop and Carboxyl Terminus of the 5-HT2A Receptor—Using synthetic peptides encompassing amino acids 183–202 (i2) and 377–396 (ct) of the 5-HT2A receptor, we tested the ability of either or both regions to interact with CaM with a modified blot overlay technique. Increasing amounts of each peptide (1–100 nmol) were slot-blotted to PVDF membranes and incubated with biotinylated CaM. To separate specific from nonspecific binding, we used a negative control peptide corresponding to the 17-amino acid CaM binding region of myosin light chain kinase, which contains a point mutation that renders it unable to bind CaM. Both the i2 and ct peptide bound biotinylated CaM in buffer containing 0.1 mm Ca2+, whereas the myosin light chain kinase control peptide showed no binding (Fig. 3). Binding was significantly reduced when Ca2+ was removed from the b