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Originally published In Press as doi:10.1074/jbc.M501696200 on June 21, 2005

J. Biol. Chem., Vol. 280, Issue 35, 30741-30750, September 2, 2005
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Interaction of Calmodulin with the Serotonin 5-Hydroxytryptamine2A Receptor

A PUTATIVE REGULATOR OF G PROTEIN COUPLING AND RECEPTOR PHOSPHORYLATION BY PROTEIN KINASE C*

Justin H. Turner and John R. Raymond{ddagger}

From the Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, and the Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, February 14, 2005 , and in revised form, June 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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]GTP{gamma}S 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The serotonin 5-hydroxytryptamine2A (5-HT2A)1 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 (1). 5-HT2A receptors are also highly expressed in vascular smooth muscle and renal mesangial cells, where they mediate contraction and proliferation (2–4), and platelets, where they contribute to aggregation and adherence (5), as well as in kidney (6) and skeletal muscle (7, 8). 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 (9–12). 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 (13–15). Other second messengers and effectors regulated by the 5-HT2A receptor include phospholipase A2 (16–18), phospholipase D (19), Ca2+ channels (20–22), reactive oxygen and nitrogen species (23, 24), and Na+/H+ exchange (25, 26). 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 (27). 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 (28, 29). 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 (30, 31). 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 {beta}{gamma} subunits (32). 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 (33, 34), whereas Nickols et al. (35) 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 (36). 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 GTP{gamma}S binding to crude membrane preparations.2 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 (37). Berg et al. (38) 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 (39, 40). Finally, the 5-HT2A receptor activates extracellular signal-regulated mitogen-activated protein kinases through the intermediate action of Ca2+/CaM (1).

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 (36). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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%20Biotechnology">Upstate Biotechnology (Charlottesville, VA). Dansyl chloride was purchased from Molecular Probes (Eugene, OR) and [35S]GTP{gamma}S 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 x 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 2x 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 (41). 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 3x 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. (42). 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 CentriconTM 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 8000TM 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 {beta}-glycerolphosphate, 1 mM dithiothreitol, 1 mM CaCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 100 µM [{gamma}-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 [{gamma}-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.



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  		FIG. 1.
CaM co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts. Wild-type NIH-3T3 cells and NIH-3T3 cells overexpressing the 5-HT2A receptor were treated with either 1 µM 5-HT or vehicle for 5 min. Cell lysates were immunoprecipitated with commercial anti-5-HT2A receptor antibody and samples were resolved with SDS-PAGE, followed by immunoblotting with an anti-CaM antibody or a second 5-HT2A receptor antibody (generous gift from Ryan Strachan and Dr. Bryan Roth, Case Western Reserve University).

 
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 x g for 10 min to remove whole cells and nuclear debris, and the supernatant was centrifuged at 37,000 x 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]GTP{gamma}S Binding to NIH-3T3/5-HT2A Receptor Cell Membranes— The protocol for measurement of [35S]GTP{gamma}S binding to crude cell membranes was adapted from the methods of Cussac et al. (43) and Adlersberg et al. (44). 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]GTP{gamma}S (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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (1) and Na+/H+ exchange (26, 45). 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 (32), µ-opioid receptors (34), and 5-HT1A receptor (36), 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 (47, 48). Predictably, identified CaM binding regions conform to short peptides that form amphipathic {alpha}-helices composed of hydrophobic and positively charged amino acid residues (49). Using a web-based computer search program, which identifies such regions based on evaluation criteria such as hydropathy, {alpha}-helical propensity, residue charge, helical class, residue weight, and hydrophobic residue content (50), 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 (50). As expected, like most CaM-binding sites, both helical wheels showed clusters of positively charged amino acids on one side of the {alpha}-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 buffer and replaced with 1 mM EGTA. Interestingly, the i2 peptide appeared to bind more readily to CaM than did the ct peptide, indicating that, although both peptides bind CaM in a Ca2+-sensitive manner, they may do so with differing affinities.



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  		FIG. 2.
The 5-HT2A receptor contains two putative CaM-binding domains. A, schematic representation of the 471-amino acid, 7-transmembrane-spanning 5-HT2A receptor. The Swiss-Prot protein sequence for the human 5-HT2A receptor was entered into a CaM-binding site search engine (calcium.uhnres.utoronto.ca/ctdb/pub_pages/search/index.htm). Scores were evaluated for a sliding 20-amino acid window and normalized for the entire sequence, with higher numbers corresponding to the most likely binding sites. The sequence search revealed two putative CaM-binding sites, one in the second intracellular loop, and one in the carboxyl terminus of the receptor. B, sequence alignments between putative 5-HT2A receptor CaM binding regions and known CaM-binding domains. Both putative 5-HT2A receptor CaM-binding domains were classified into motif groups based on sequence similarity to other CaM-binding proteins. The second intracellular loop domain was classified as a 1-8-14 motif, marked by key hydrophobic residues at positions 1, 8, and 14. The sequence was aligned with other 1-8-14 motifs, including those for the neuronal and endothelial nitric oxide synthases. In contrast, the carboxyl-terminal domain was classified as a 1-10 motif, characterized by hydrophobic residues at positions 1 and 10. The sequence was aligned with other 1-10 motifs, including those for kinesin-like CaM-binding protein and phosphofructokinase.

 
We next analyzed the peptide-CaM complexes using polyacrylamide gel electrophoresis in the presence of 4 M urea. This technique eliminates lower affinity and nonspecific protein-protein interactions (KD > 100 nM). Increasing amounts of peptide (75–3000 pmol) were incubated with a constant amount of CaM (300 pmol) and subsequently analyzed by non-denaturing gel electrophoresis. In the presence of Ca2+, the addition of the 5-HT2A i2 peptide produced an upward shift in the migration of CaM (Fig. 4A). In contrast, no peptide-CaM complexes were formed when Ca2+ was chelated with EGTA. The ct peptide produced no readily apparent shift in the mobility of CaM (Fig. 4B). This suggests that the ct peptide probably binds CaM with an affinity weaker than 100 nM, however the density of the CaM band decreased consistently with increasing peptide concentration, indicating that some shift may have occurred without detection. The incomplete gel shift induced by the i2 peptide, even at a peptide-CaM molar ratio of 10:1, is in line with published reports of other CaM binding regions that utilized this method under the same conditions (33, 36, 52). Likewise, incubation with a peptide derived from the CaM binding region of myosin light chain kinase, which binds CaM with very high affinity (~6 pM), was also unable to induce a complete shift in CaM mobility in our hands (Fig. 4C).

Interaction of 5-HT2A i2 and ct Peptides with Dansyl-CaM— Precise affinities are often difficult to calculate using gel-shift assays or in vitro binding techniques. Thus, we evaluated the binding affinities of the i2 and ct peptides for CaM by measuring changes in the fluorescence emission spectrum of dansyl-CaM. Ligand binding to dansyl-CaM enhances the fluorescence emission and blue-shifts the emission peak to a lower wavelength. As shown in Fig. 5A, dansyl-CaM displays weak fluorescence in the absence of Ca2+, with an emission peak of ~520 nm. In the presence of Ca2+, the fluorescence emission is enhanced and shifted to a lower wavelength. As expected, both i2 and ct peptides further enhanced dansyl-CaM fluorescence and shifted the emission peak to ~495 nm, whereas the negative control myosin light chain kinase mutant peptide was completely ineffective (data not shown). The amounts of i2 and ct peptides were then varied to generate typical concentration-dependent saturation curves. The fluorescence emission spectra of dansyl-CaM (0.1, 0.2, 0.3, 0.4, and 0.5 µM) were measured in the presence of increasing concentrations of i2 or ct peptides (0–2 µM), and resulting data points were fit by nonlinear least squares analysis to the Scatchard one-site binding equation (Fig. 5, B and C). As expected, higher concentrations of dansyl-CaM required increased concentrations of peptide to enhance fluorescence and reach saturation, resulting in variations in EC50 measurements. This results from a depletion of free peptide, a phenomenon which is most apparent at low concentrations of dansyl-CaM. By calculating EC50 values over a range of dansyl-CaM concentrations it was possible to extrapolate to an infinitely low dansyl-CaM concentration, at which point depletion is essentially nonexistent. As shown in Fig. 5D and 5E, EC50 measurements derived from peptide binding curves were plotted against the concentration of dansyl-CaM. These values fell onto straight lines with slopes of close to one, indicative of a 1:1 binding stoichiometry. Extrapolating the lines to the y-axis gave "true affinities" of 65 ± 30 nM and 168 ± 38 nM for the i2 and ct peptides, respectively.



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  		FIG. 3.
Interactions of 5-HT2A receptor synthetic peptides with biotinylated CaM. Increasing amounts (1, 3, 10, 30, and 100 nmol) of peptides were slot-blotted to PVDF membrane and subjected to overlay with 0.5 µg/ml biotinylated CaM in the presence of either 0.1 mM CaCl2 or 1 mM EGTA as described under "Experimental Procedures." Bound biotinylated CaM was then detected with alkaline phosphatase-conjugated avidin and a chemiluminescent reagent. Biotinylated CaM bound to both the i2 and ct peptides, but not to a myosin light chain kinase-negative control peptide, in the presence of Ca2+. CaM binding was significantly reduced in the presence of EGTA. Data are representative of three separate experiments.

 



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  		FIG. 4.
Complex formation between CaM and 5-HT2A receptor synthetic peptides. CaM (300 pmol) was incubated with increasing amounts (0, 75, 150, 300, 600, 1500, and 3000 pmol) of peptide in the presence of 4 M urea and either 0.1 mM CaCl2 or 1 mM EGTA. Complexes were then resolved by nondenaturing polyacrylamide gel electrophoresis, in the presence of 4 M urea and either CaCl2 or EGTA. Gels were stained with GelCodeBlue staining reagent. The i2 peptide (A) and the authentic myosin light chain kinase peptide (C), but not the ct peptide (B), caused a shift in the migration of CaM in the presence of Ca2+, but not in the presence of EGTA. Data are representative of three separate experiments.

 
Functional Overlap of CaM-binding Domain and PKC Phosphorylation Site within the 5-HT2A Receptor ct Peptide— Most GPCRs can be modulated by kinase-directed phosphorylation of threonine and serine residues, with the second messenger-dependent kinases (PKA, PKC, and others) predominating at low agonist concentrations, and the G protein-coupled receptor kinases predominating at high agonist concentrations and over extended periods of time (53). Although direct phosphorylation of the 5-HT2A receptor has yet to be reported, both 5-HT2A receptor internalization and desensitization appear to be highly dependent on PKC activation (54, 55). The 5-HT2A receptor contains six putative PKC phosphorylation sites, five of which are localized to the receptor third intracellular loop. Interestingly, the only putative PKC phosphorylation site in the carboxyl terminus (384NKTYR385) is centrally located within the ct CaM binding region (Fig. 6A). We consequently wondered whether binding of CaM to this region might regulate phosphorylation of this residue by PKC and, conversely, whether phosphorylation of this residue might inhibit CaM binding. Because typical CaM binding regions contain few or no acidic (negatively charged) amino acids, phosphorylation of the target threonine residue in the ct CaM binding region would add a negative charge and would consequently be expected to perturb CaM binding. We tested this hypothesis by evaluating the fluorescence emission spectrum of dansyl-CaM in the presence of a synthetic peptide identical to the ct peptide, but which contains a phosphorylated threonine at residue 386 (ct-P). As shown in Fig. 6B, the increase in fluorescence induced by the phosphorylated peptide was highly diminished as compared with the shift induced by the unphosphorylated peptide, suggesting that phosphorylation of the ct peptide inhibits CaM binding.

We next assessed whether the peptide could actually be phosphorylated by PKC in vitro, and if so, whether this phosphorylation could be inhibited by CaM. Purified rat brain PKC (composed primarily of PKC-{alpha} and PKC-{beta} isozymes) readily phosphorylated the ct peptide (Km = 8.83 ± 2.31 µM, Vmax = 48.85 ± 7.94 nmol/min/mg). In contrast, phosphorylation of the 5-HT2A receptor i2 peptide was not observed under identical conditions (data not shown). Interestingly, phosphorylation of the ct peptide was dose-dependently decreased in the presence of CaM, with a 20 µM concentration of CaM able to inhibit ct phosphorylation by ~80%. These data suggest that phosphorylation of the ct peptide by PKC and peptide binding to CaM are likely to be mutually inhibitory.



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  		FIG. 5.
Interaction of i2 and ct with dansyl-CaM. The fluorescence emission spectra of dansyl-CaM were measured at an excitation wavelength of 340 nm. A, traces represent the fluorescence emission measured from 400 to 600 nm for dansyl-CaM (0.5 µM) alone, in the presence of 0.1 mM CaCl2, and in the presence of 0.1 mM CaCl2 plus i2 or ct peptides (2 µM). B and C, concentration-dependent enhancement of dansyl-CaM fluorescence was measured at an emission wavelength of 495 nm in the presence of 0.1 µM CaCl2. Dansyl-CaM (0.1, 0.2, 0.3, 0.4, and 0.5 µM) was incubated with increasing concentrations of i2 or ct peptides (0–2 µM), and measurements were plotted by nonlinear least squares analysis using the Boltzmann one-site binding equation. D and E, apparent KD measurements calculated from binding curves were plotted versus dansyl-CaM concentration using the Hill equation by curvilinear regression to determine the true affinities. Data represent means ± S.E. for three separate experiments performed at each concentration of dansyl-CaM.

 
CaM Inhibits 5-HT2A Receptor Coupling to G Proteins—The 5-HT2A receptor modulates the activity of numerous second messengers and effectors, primarily through the {alpha} subunits of Gq/11 heterotrimers. Although the G protein contact sites of the 5-HT2A receptor have yet to be detailed experimentally, the G protein-coupling sites of other GPCRs have been mapped to diverse regions of the second intracellular loop, third intracellular loop, and carboxyl terminus (56–60). Binding of CaM to intracellular regions of the 5-HT2A receptor might be expected to sterically hinder receptor access to G protein subunits. This appears to be the case with some Gi/o-coupled receptors, including the D2 dopamine (33) and µ-opioid (34) receptors, both of which interact with CaM via their extended third intracellular loops. However, a similar observation has yet to be reported for Gq/ll-coupled receptors. We assessed 5-HT2A receptor coupling to heterotrimeric G proteins by measuring the binding of nonhydrolyzable [35S]GTP{gamma}S to cell membranes. For these experiments we used NIH-3T3 cells, which significantly overexpress the 5-HT2A receptor. The use of an artificial receptor expression system was essential to achieve an adequate signal-to-noise ratio, due largely to the fact that G{alpha}q/ll proteins are relatively low in abundance, and have much lower rates of nucleotide exchange than G{alpha}i/o proteins. Stimulation of NIH-3T3 membranes with 10 µM 5-HT produced an ~25% increase in [35S]GTP{gamma}S binding (Fig. 7A). Interestingly, CaM dose-dependently decreased [35S]GTP{gamma}S binding, with concentrations of 1–10 µM reducing [35S]GTP{gamma}S binding to basal levels or lower. This effect was not due to nonspecific effects of the CaM protein, in that membranes incubated with the Ca2+-binding protein S-100 (which has a similar molecular weight to CaM but different target binding proteins) did not attenuate the 5-HT-induced increase in [35S]GTP{gamma}S binding (data not shown). These data suggest that interaction of CaM with the 5-HT2A receptor may regulate receptor coupling to heterotrimeric G proteins. However, it remained unclear whether this effect was via CaM binding to the second intracellular loop, carboxyl terminus, or both regions of the receptor.



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  		FIG. 6.
CaM as a putative regulator of 5-HT2A receptor phosphorylation by PKC. A, putative PKC phosphorylation site in the 5-HT2A receptor ct peptide. B, change in fluorescence emission of dansyl-CaM in the presence of ct and ct-P peptides. Bars represent the change in fluorescence emission measured at 495 nm for dansyl-CaM (0.5 µM) alone and in the presence of ct (2 µM) or ct-P peptide (2 µM). C, inhibition of PKC-mediated ct peptide phosphorylation by CaM. Increasing concentrations of ct peptide (0–6 µM) in the presence of varying concentrations of CaM (0, 4, 10, and 20 µM) were incubated with [{gamma}-32P]ATP and purified rat brain PKC for 1 h at 30 °C, and incorporated radioactivity was measured as described under "Experimental Procedures." Data are expressed as the percentage of maximum phosphorylation of peptide. Results shown represent the mean ± S.E. for at least three experiments.

 
Previous studies have shown that synthetic peptides derived from specific receptor-G protein interface sites can mimic the activated receptors themselves. This methodology has been used extensively to characterize G protein interaction domains for multiple receptors, including those for angiotensin II (60), glucagon-like peptide (57), and dopamine (61). We investigated the ability of the 5-HT2A receptor CaM-binding domains to stimulate G protein coupling by measuring [35S]GTP{gamma}S binding to NIH-3T3 cell membranes in the presence of increasing concentrations of i2 or ct peptides. As shown in Fig. 7B, the i2 peptide strongly stimulated [35S]GTP{gamma}S binding, whereas the ct peptide had no effect. These data suggest that the second intracellular loop of the 5-HT2A receptor is largely responsible for receptor coupling to heterotrimeric G proteins. Likewise, it suggests that the ability of CaM to inhibit G protein coupling is likely mediated by binding to the high affinity CaM-binding domain in the second intracellular loop, rather than to the lower affinity site in the carboxyl terminus.



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  		FIG. 7.
Effect of CaM on 5-HT2A receptor coupling to heterotrimeric G proteins. A, [35S]GTP {gamma}S binding to cell membranes derived from NIH-3T3 cells overexpressing the 5-HT2A receptor (generous gift from Dr. Elaine Sanders-Bush, Vanderbilt University) was evaluated in the absence and presence of increasing concentrations of CaM (0.1–10 µM). GTP{gamma}S binding was determined in the presence of 50 µM GDP, with or without 10 µM 5-HT, and expressed as a percentage of basal binding in unstimulated cells. Results shown represent the mean ± S.E. (n = 6, *, p < 0.05) versus control cell membranes. B, [35S]GTP{gamma}S binding to cell membranes derived from NIH-3T3 cells was evaluated in the absence and presence of increasing concentrations of i2 or ct peptides (0.1–10 µM). Data are expressed as percentage of basal binding in unstimulated cells (n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
What is new about this work is that we have characterized two putative CaM-binding domains in the 5-HT2A receptor, and have shown that the interaction of CaM with these domains likely attenuates G protein coupling to the second intracellular loop and phosphorylation of the carboxyl terminus of the receptor by PKC. To our knowledge, this is the first demonstration that CaM attenuates G protein coupling of a G{alpha}q/11-preferring receptor. We showed that CaM co-immunoprecipitates with the 5-HT2A receptor from NIH-3T3 fibroblasts and that the amount of CaM co-immunoprecipitating with the receptor was increased after treatment with 5-HT, suggesting that CaM association is dependent on receptor activation. We subsequently identified two putative CaM-binding sites located within the second intracellular loop and carboxyl terminus of the receptor. These sites have properties typical of CaM-binding domains, including putative amphipathic {alpha}-helical structures composed of basic and hydrophobic amino acid residues. In addition, both sites could be aligned with well established CaM-binding motifs based on the locations of key hydrophobic residues. Synthetic peptides derived from the i2 and ct regions bound to CaM in a Ca2+-sensitive manner with average KD values of ~65 and ~168 nM, respectively. Interestingly, many GPCRs appear to contain one or more putative CaM-binding sites, suggesting that interaction of CaM with different receptor regions may represent a common mechanism through which GPCR-dependent signaling pathways may be regulated (Table I).


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  		TABLE I
Location of putative CaM binding regions in GPCR families

The Swiss-Prot protein sequences for members of the human serotonin, adrenergic, and muscarinic GPCR families were entered into a CaM binding site search engine (calcium.uhnres.utoronto.ca/ctdb/pub_pages/search/index.htm). Putative CaM-binding motifs were identified in juxtamembrane regions of the second intracellular loops, third intracellular loops, and/or carboxyl termini of the receptors. As evidenced in the table, the presence and location of putative CaM-binding motifs is a common characteristic of GPCRs and is largely receptor subtype-dependent.

 
The functions of the 5-HT2A receptor CaM-binding domains have not been completely elucidated. However, these regions have previously been implicated in numerous signaling pathways and processes. The importance of the second intracellular loop in G protein coupling has been experimentally confirmed for other GPCRs, including the bradykinin B2 (62), {alpha}2A adrenergic (63), and type B endothelin receptors (64). Using molecular modeling, Prado et al. (62) produced evidence that the distal i2 loop and proximal carboxyl terminus of the bradykinin B2 receptor may interact with each other to mediate receptor internalization and resensitization, in addition to G protein coupling. Berg et al. (65) showed that the i2 loop of the 5-HT2C receptor is important for G{alpha}q coupling, and that different isoforms of the receptor produced via RNA editing in regions of the second intracellular loop varied in their ability to induce second messengers.

Previous work has demonstrated that CaM binding to the receptor third intracellular loops can attenuate the interactions between G{alpha}i and several receptors, including the 5-HT1A receptor (36), µ-opioid receptor (34), D2 dopamine receptor (33), and the group III metabotropic glutamate receptor, mGluR7a (66, 67). New evidence in the current report suggests that CaM binding to the second intracellular loop of the 5-HT2A receptor likely modulates coupling to heterotrimeric G proteins, most likely G{alpha}q/11 and/or G{alpha}12/13 subunits. The affinity of the second intracellular loop of the 5-HT2A receptor for CaM (65 ± 30 nM; Fig. 6) is consistent with the ability of 1–10 µM CaM to completely attenuate 5-HT2A receptor-mediated increases in [35S]GTP{gamma}S binding (Fig. 7) and supports the possibility that bona fide interactions of CaM with the 5-HT2A receptor can occur within the context of the cell. Thus, CaM is likely to play a role in attenuating signal initiation from the 5-HT2A receptor second intracellular loop by dampening interactions with heterotrimeric G proteins.

One general mechanism of receptor desensitization involves phosphorylation of intracellular residues by second messengers such as PKC, PKA, and the G protein-coupled receptor kinases. Our group previously showed that CaM binding to synthetic peptides derived from the 5-HT1A receptor third intracellular loop could inhibit phosphorylation by protein kinase C (36). Interestingly, the 5-HT2A receptor carboxyl terminus CaM binding region contains a consensus PKC phosphorylation site, 384NKTYR388. It is well established that the 5-HT2A receptor can activate PKC via the hydrolysis of phosphatidylinositol bisphosphate and the subsequent formation of diacylglycerol. Berg et al. (55) showed that agonist-induced desensitization of the 5-HT2A receptor was dependent on the activity of PKC and could be modulated by pharmacological inhibitors of CaM. Likewise, activation of PKC in HEK293 cells induced the internalization and recycling of a 5-HT2A receptor green fluorescent protein fusion protein (54). The current work also supports a potential role for CaM in modulating phosphorylation of the 5-HT2A receptor. We showed for the first time that the putative PKC phosphorylation site in the 5-HT2A receptor carboxyl terminus is an excellent PKC substrate in vitro. Furthermore, CaM was capable of concentration-dependently decreasing the ability of PKC to phosphorylate a peptide derived from the ct region. The apparent agonist-dependent binding of CaM to the 5-HT2A receptor carboxyl terminus suggests a possible mechanism by which this receptor may be partially protected from PKC-dependent phosphorylation and perhaps subsequent internalization and desensitization. Furthermore, we speculate that the failure of several groups to demonstrate PKC-mediated phosphorylation of the 5-HT2A receptor in vivo may be due, in part, to steric blockade of PKC sites by CaM in live cells. This suggests that a complex contribution of both CaM and PKC may regulate 5-HT2A receptor internalization and desensitization.

Mutation of Ser188 in the second intracellular loop has been shown to attenuate 5-HT2A receptor desensitization (68). Interestingly, this residue is predicted to be a phosphorylation site for the dual-specificity kinase Clk-2. The S188A mutation also caused a 3-fold increase in the EC50 and a 40% reduction in 5-HT efficacy, suggesting that post-translational modification and/or accessory protein binding to this region might directly disrupt G protein coupling, leading to internalization-independent desensitization. The second intracellular loop of the 5-HT2A receptor also contains a DRYXX(I/V)XXP motif similar to that identified in the human muscarinic cholinergic receptor, which has been proposed as an important regulator of receptor internalization, whereas the membrane-proximal region of the 5-HT2A receptor carboxyl terminus has tyrosine-containing motifs previously shown to mediate internalization of GPCRs for neurokinin 1 (69), parathyroid hormone (70), and angiotensin II (71). Such tyrosine-based motifs have been shown to form clathrin-associated protein complexes. CaM has been found to play a role in the general process of receptor endocytosis and trafficking in yeast (72) and human epithelial cells (73). Our group reported a role for CaM in 5-HT1A receptor internalization, a required step for MEK and subsequent ERK activation (74). Conversely, pharmacological inhibitors of CaM were found to inhibit recycling and degradation of the EGF receptor without affecting internalization, resulting in the accumulation of receptors in enlarged endosomal structures (75). Thus, it is conceivable that CaM could regulate 5-HT2A receptor internalization and/or trafficking through direct binding to the receptor.

The current work has significance that extends beyond the 5-HT receptor family in that many GPCRs possess putative CaM binding domains (Table I) in intracellular juxtamembrane regions of the second and third intracellular loops and carboxyl termini. Thus far, these motifs have been implicated in G protein coupling and PKC-induced phosphorylation of both G{alpha}i- and G{alpha}q/11-coupled receptors, for which CaM appears to dampen both processes. Because CaM has been implicated in various other GPCR processes such as ERK activation, stimulation of Na+/H+ exchange, and receptor trafficking (1, 26, 72–77), direct binding of CaM to GPCRs could modulate any number of receptor processes. Additionally, the presence of putative CaM binding motifs in Gs-coupled receptors such as the {beta}1-, {beta}2-, and {beta}3-adrenergic and 5-HT4 and 5-HT7 receptors (Table I) raises the possibility that CaM might influence cAMP accumulation by directly binding to those receptors, although that possibility remains to be verified experimentally.

In conclusion, we have identified for the first time the presence of CaM-binding sites in the 5-HT2A receptor. These sites reside in juxtamembrane regions of the second intracellular loop and carboxyl terminus of the receptor. To our knowledge, this represents the first example of a GPCR with a CaM-binding site in the second intracellular loop and the first demonstration that CaM binding can attenuate G protein coupling to a G{alpha}q/11-preferring receptor. In addition, the work extends the concept that CaM binding to GPCR can inhibit receptor phosphorylation by PKC. These results suggest that CaM may serve as a valuable regulator and/or second messenger of 5-HT2A receptor function.


   FOOTNOTES
 
* This work was supported by Department of Veterans Affairs Merit and Research Enhancement Award Program awards (to J. R. R.), by National Institutes of Health Grants GM08716 (to J. H. T.) and DK52448 and GM63909 (to J. R. R.), by a predoctoral fellowship from the American Heart Association, Mid-Atlantic Affiliate (Grant 0215195U to J. H. T.), and by laboratory endowments jointly supported by the Medical University of South Carolina, Division of Nephrology and Dialysis Clinics, Inc. (to J. R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Medical University of South Carolina, 96 Jonathan Lucas St., Rm. 829 CSB, P. O. Box 250623, Charleston, SC 29425-2227. Tel.: 843-876-5128; Fax: 843-876-5129; E-mail: raymondj{at}musc.edu.

1 The 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; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; ct-P, a synthetic peptide identical to the ct peptide but containing a phosphorylated threonine at residue 386. Back

2 J. H. Turner and J. R. Raymond, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Elaine Sanders-Bush (Nashville, TN) for kindly providing NIH-3T3 cells overexpressing the 5-HT2A receptor and for providing excellent advice. We also thank Ryan Strachan (Cleveland, OH) and Dr. Bryan Roth (Cleveland, OH) for kindly providing rabbit polyclonal anti-5-HT2A receptor antibodies.



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