Faculty Collaboration Database

Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C. J Biol Chem 2004 Apr 23;279(17):17027-37

Date

01/31/2004

Pubmed ID

14752100

DOI

10.1074/jbc.M313919200

Scopus ID

2-s2.0-2342425115 (requires institutional sign-in at Scopus site)   75 Citations

Abstract

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 Ca(2+)-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 Ca(2+)-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 approximately 110 nm for the i3N peptide and approximately 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 microm. 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.

Author List

Turner JH, Gelasco AK, 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 Motifs
Amino Acid Sequence
Animals
Binding Sites
Biotinylation
Brain
CHO Cells
Calmodulin
Cattle
Cricetinae
Dose-Response Relationship, Drug
Fibroblasts
Fluorescence Resonance Energy Transfer
Fluorometry
Kinetics
Molecular Sequence Data
Peptides
Phosphorylation
Plasmids
Precipitin Tests
Protein Binding
Protein Conformation
Protein Kinase C
Protein Structure, Secondary
Protein Structure, Tertiary
Rats
Receptor, Serotonin, 5-HT1A
Sequence Homology, Amino Acid
Signal Transduction
Spectrometry, Fluorescence
Surface Plasmon Resonance
Time Factors
Transfection