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 behavior. 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 (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, alpha-MSH-ND was the most efficient alpha-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of alpha-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor-ligand complex by NMR, [Gln6]alpha-MSH-ND and [Lys6]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 different.
Lee
E J
EJ
Laboratory of Molecular Biology, Medical Research Center, Department of Pharmacology, College of Medicine, Brain Korea 21 Project for Medical Sciences, Seoul, South Korea.
Lee
S H
SH
Jung
J W
JW
Lee
W
W
Kim
B J
BJ
Park
K W
KW
Lim
S K
SK
Yoon
C J
CJ
Baik
J H
JH
D000324:Adrenocorticotropic Hormone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements; D013997:Time Factors; D014158:Transcription, Genetic; D000521:alpha-MSH / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D008024:Ligands; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin; D000521:alpha-MSH; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D008156:Luciferases