Faculty Collaboration Database

Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C. J Biol Chem 2005 Sep 02;280(35):30741-50

Date

06/23/2005

Pubmed ID

15970592

DOI

10.1074/jbc.M501696200

Scopus ID

2-s2.0-24744463670 (requires institutional sign-in at Scopus site)   59 Citations

Abstract

The 5-hydroxytryptamine2A (5-HT2A) 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-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 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-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPgammaS 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.

Author List

Turner JH, Raymond JR

Author

John R. Raymond MD President, CEO, Professor in the President department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Binding Sites
Calmodulin
Cattle
Dansyl Compounds
Fibroblasts
Guanosine 5'-O-(3-Thiotriphosphate)
Heterotrimeric GTP-Binding Proteins
Humans
Mice
Molecular Sequence Data
NIH 3T3 Cells
Peptides
Protein Binding
Protein Conformation
Protein Kinase C
Rats
Receptor, Serotonin, 5-HT2A
Sequence Alignment
Serotonin