Differential regulation of cAMP-mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors

Melanocortins are known to be involved in the regulation of feeding behavio r. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC 3R) in HEK 293T cells, by using a luciferase reporter gene under the transc riptional control of a cAMP-responsive element (CRE) as a monitor of intrac ellular cAMP levels and cAMP-regulated gene expression. We were able to sho w that MC4R and MC3R expressed in the human cell line HEK 293T stimulate tr anscription induced by stimulation with different analogs of ol-melanocyte- stimulating hormone (alpha -MSH) at different levels. In our assay of CRE-m ediated gene transcription activity, alpha -MSH-ND was the most efficient a lpha -MSH analog for; MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of alpha -MSH-ND to Gin or Lys markedly decrease d CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor-ligand complex by NMR, [Gln6]alpha -MSH-ND and [L ys6]alpha -MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30-50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor -ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively diffe rent.