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J. Biol. Chem., Vol. 280, Issue 35, 30741-30750, September 2, 2005

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Interaction of Calmodulin with the Serotonin 5-Hydroxytryptamine_2A Receptor

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

Justin H. Turner and John R. Raymond

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

The 5-hydroxytryptamine_2A (5-HT_2A) receptor is a G_q/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-HT_2A 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-HT_2A 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 K_D 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-HT_2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT_2A receptor-mediated [³⁵S]GTPS 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-HT_2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function.

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

^* 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.

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 .

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