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Papers In Press, published online ahead of print January 29, 2004
Medicine, Medical University of South Carolina, Charleston, SC 29425
Corresponding Author: raymondj{at}musc.edu
The serotonin 5-HT1A receptor couples to heterotrimeric G-proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor interacting proteins (RIPs) in 5-HT1A receptor signaling. We have found that the ubiquitous Ca2+-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT1A receptor in Chinese hamster ovary (CHO) fibroblasts. The human 5-HT1A 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 KD 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
J. Biol. Chem, 10.1074/jbc.M313919200
Submitted on December 19, 2003
Revised on January 26, 2004
Accepted on January 29, 2004
Calmodulin interacts with the third intracellular loop of the serotonin 5-HT(sub1A) receptor at two distinct sites. Putative role in receptor phosphorylation by protein kinase C
M. We used confocal microscopy and bioluminescence resonance energy transfer (BRET) to show that CaM interacts with the 5-HT1A 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-HT1A receptor i3 loop peptides by protein kinase C (PKC) are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT1A receptor phosphorylation and desensitization. These data suggest that the 5-HT1A receptor contains high and moderate affinity CaM-binding regions that may play important roles in receptor signaling and function.
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