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

A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca2+ indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [I-123] 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 EC50 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. (C) 2001 Elsevier Science.