Calmodulin Interacts with the Third Intracellular Loop of the Serotonin 5-Hydroxytryptamine1A Receptor at Two Distinct Sites

Abstract

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 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 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 μm. We used bioluminescence resonance energy transfer 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 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.