Calmodulin interaction with peptides from G-protein coupled receptors measured with S-Tag labeling

Biochem Biophys Res Commun. 2005 Jul 29;333(2):390-5. doi: 10.1016/j.bbrc.2005.05.129.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Calmodulin / chemistry*
  • Enzyme-Linked Immunosorbent Assay / methods
  • Humans
  • Peptide Fragments / analysis
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptides / chemistry
  • Protein Binding
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / genetics
  • Recombinant Fusion Proteins / chemistry
  • Ribonucleases / analysis
  • Ribonucleases / chemistry*
  • Ribonucleases / genetics
  • Staining and Labeling / methods

Substances

  • Calmodulin
  • Peptide Fragments
  • Peptides
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • ribonuclease S-peptide
  • Ribonucleases