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 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.
Carreras
Christopher W
CW
Department of Pharmacological Sciences, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA. carreras@kosan.com
Siani
Michael A
MA
Santi
Daniel V
DV
Dillon
Susan B
SB
D000331:Aequorin / Q000737:chemistry*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D008163:Luminescent Measurements; D009037:Motilin / Q000378:metabolism / Q000494:pharmacology; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists / Q000235:genetics* / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors
D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D000331:Aequorin; D009037:Motilin