title: 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.
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.
authors.affiliation: Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina, USA.
authors.forename: Justin H
authors.initials: JH
authors.lastname: Turner
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Gelasco
authors.forename: John R
authors.initials: JR
authors.lastname: Raymond
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D044282:Receptor, Serotonin, 5-HT1A / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection
chemical_list: C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D002147:Calmodulin; D010455:Peptides; D044282:Receptor, Serotonin, 5-HT1A; D011493:Protein Kinase C