Stable Expression of a Synthetic Gene for the Human Motilin Receptor: Use in an Aequorin-Based Receptor Activation Assay
✍ Scribed by Christopher W. Carreras; Michael A. Siani; Daniel V. Santi; Susan B. Dillon
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 88 KB
- Volume
- 300
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
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 dosedependent 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.