A Functional Assay for MC1R Mediated Gs–coupled Signaling Pathways

Abstract

Abstract: : Purpose: We aim at developing an assay to study melanocortin–1 receptor (MC1R) function through intracellular signal pathway,which will lay the foundation of MC1R mechanism in retinal and retinal related disease. Methods: We cloned MC1R gene from human genomic DNA. Then we cotransfected 4xCRE/EGFP vector and MC1R into CHO cells and selected clones with G418. MC1R expression was detected by northern blot and western blot. Cyclic AMP is evaluated by the expression of EGFP with forskolin.We test the function of MC1R with alpha–MSH and AGRP as standard agonist and antagonist. Results: We have cloned MC1R gene successfully.Both northern blot and western blot confirmed the expression of MC1R in CHO cells.EGFP expression can be detected 6 hours after addition of forskolin or alpha–MSH and can not be detected when treated with AGRP. Conclusions: Current assays for activation of Gs–coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. We have developed a rapid nonradioactive assay utilizing EGFP as report gene that fused to four copies of the cyclic AMP response element(4xCRE) to detect the function of MC1R that results in an increase or decrease of intracellular cAMP. All the experiments indicate that the assay is sensitive and convenient. This assay is also useful for the characterization of other Gs–coupled receptors.

Keywords: retina • retinal pigment epithelium