Stable expression of a synthetic gene for the human motilin receptor: use in an aequorin-based receptor activation assay

Anal Biochem. 2002 Jan 15;300(2):146-51. doi: 10.1006/abio.2001.5466.

Abstract

A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K(d) value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC(50) values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1-14) inhibited motilin induced response with a K(i) value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.

MeSH terms

  • Aequorin / chemistry*
  • Biological Assay / methods*
  • Cell Line
  • Gene Expression*
  • Genetic Engineering*
  • Humans
  • Luminescent Measurements
  • Motilin / metabolism
  • Motilin / pharmacology
  • Receptors, Gastrointestinal Hormone / agonists
  • Receptors, Gastrointestinal Hormone / genetics*
  • Receptors, Gastrointestinal Hormone / metabolism*
  • Receptors, Neuropeptide / agonists
  • Receptors, Neuropeptide / genetics*
  • Receptors, Neuropeptide / metabolism*
  • Time Factors

Substances

  • Receptors, Gastrointestinal Hormone
  • Receptors, Neuropeptide
  • motilin receptor
  • Aequorin
  • Motilin