Calmodulin interaction with peptides from G-protein coupled receptors measured with S-Tag labeling.
We have developed a quantitative assay of calmodulin (CaM) binding to S-Tag labeled peptides derived from G-protein coupled receptor (GPCR) sequences. CaM binding of peptides derived from the third intracellular loop (i3) of mu opioid receptor (MOR) was confirmed and the CaM-binding motif refined. A MORi3 peptide with a Lys > Ala substitution--shown to reduce CaM-binding of intact MOR--bound fivefold less avidly than the wild-type peptide. Screening peptides derived from i3 loops of other GPCR families confirmed 5HT1A, and identified muscarinic receptor 3, and melanocortin receptor 1, as proteins carrying CaM-binding domains. The use of S-Tag labeling can serve for rapid screening of putative CaM-binding domains in GPCRs.
Zhang
Ying
Y
Department of Pharmacology, College of Medicine and Public Health, Ohio State University, Columbus, OH 43210, USA.
Wang
Danxin
D
Sadée
Wolfgang
W
D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods
D002147:Calmodulin; D010446:Peptide Fragments; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C023560:ribonuclease S-peptide; D012260:Ribonucleases