Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C.
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.
Turner
Justin H
JH
Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425-2227, USA.
Raymond
John R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003619:Dansyl Compounds / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051381:Rats; D044402:Receptor, Serotonin, 5-HT2A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012701:Serotonin / Q000378:metabolism*
D002147:Calmodulin; D003619:Dansyl Compounds; D010455:Peptides; D044402:Receptor, Serotonin, 5-HT2A; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D020962:Heterotrimeric GTP-Binding Proteins