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J. Biol. Chem., Vol. 279, Issue 17, 17027-17037, April 23, 2004

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Calmodulin Interacts with the Third Intracellular Loop of the Serotonin 5-Hydroxytryptamine_1A Receptor at Two Distinct Sites

PUTATIVE ROLE IN RECEPTOR PHOSPHORYLATION BY PROTEIN KINASE C^*

Justin H. Turner, Andrew K. Gelasco, and John R. Raymond¶

From the Medical and Research Services of the 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 serotonin 5-HT_1A receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT_1A receptor signaling. We have found that the ubiquitous Ca²⁺-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT_1A receptor in Chinese hamster ovary fibroblasts. The human 5-HT_1A receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca²⁺-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K_D of 110 nM for the i3N peptide and 700 nM for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 ± 23 nM and 1.70 ± 0.16 µM. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT_1A receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT_1A receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT_1A receptor phosphorylation and desensitization. These data suggest that the 5-HT_1A receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.

Received for publication, December 19, 2003 , and in revised form, January 26, 2004.

^* This work was supported by grants from the Department of Veterans Affairs (Merit Award to J. R. R.), the National Institutes of Health Grants DK52448 and GM63909 (to J. R. R) and DK59950 and MH64795 (to A. K. G.), a pre-doctoral fellowship from the American Heart Association, Mid-Atlantic Affiliate (Grant 0215195U to J. H. T.), and laboratory endowments jointly supported by the MUSC 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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