title: Agouti-related protein (83-132) is a competitive antagonist at the human melanocortin-4 receptor: no evidence for differential interactions with pro-opiomelanocortin-derived ligands.
abstract: Interactions between pro-opiomelanocortin (POMC)-derived peptides, agouti-related protein (AGRP) and the melanocortin-4 receptor (MC4-R) are central to energy homeostasis. In this study we have undertaken comprehensive pharmacological analysis of these interactions using a CHOK1 cell line stably transfected with human MC4-R. Our main objectives were (1) to compare the relative affinities and potencies of POMC-derived peptides endogenously secreted within the hypothalamus, (2) to investigate the potency of AGRP(83-132) antagonism with respect to each POMC-derived peptide and (3) to determine whether AGRP(83-132) and POMC-derived peptides act allosterically or orthosterically. We have found that beta melanocyte-stimulating hormone (betaMSH), desacetyl alpha MSH (da-alphaMSH) and adrenocorticotrophic hormone all have very similar affinities and potencies at the MC4-R compared with the presumed natural ligand, alphaMSH. Moreover, even MSH precursors, such as beta lipotrophic hormone, showed significant binding and functional activity. Therefore, many POMC-derived peptides could have important roles in appetite regulation and it seems unlikely that alphaMSH is the sole physiological ligand. We have shown that AGRP(83-132) acts as a competitive antagonist. There was no significant difference in the potency of inhibition by AGRP(83-132) or agouti(87-132) at the MC4-R, regardless of which POMC peptide was used as an agonist. Furthermore, we have found that AGRP(83-132) has no effect on the dissociation kinetics of radiolabelled Nle4,D-Phe7 MSH from the MC4-R, indicating an absence of allosteric effects. This provides strong pharmacological evidence that AGRP(83-132) acts orthosterically at the MC4-R to inhibit Gs-coupled accumulation of intracellular cAMP.
authors.affiliation: School of Biological Sciences, University of Manchester, Stopford Building, Manchester M13 9PT, UK.
authors.forename: L E
authors.initials: LE
authors.lastname: Pritchard
authors.forename: D
authors.initials: D
authors.lastname: Armstrong
authors.forename: N
authors.initials: N
authors.lastname: Davies
authors.forename: R L
authors.initials: RL
authors.lastname: Oliver
authors.forename: C A
authors.initials: CA
authors.lastname: Schmitz
authors.forename: J C
authors.initials: JC
authors.lastname: Brennand
authors.forename: G F
authors.initials: GF
authors.lastname: Wilkinson
authors.forename: A
authors.initials: A
authors.lastname: White
mesh_terms: D054369:Agouti-Related Protein; D000818:Animals; D001069:Appetite Regulation; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology*; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D054369:Agouti-Related Protein; D010446:Peptide Fragments; D044105:Receptor, Melanocortin, Type 4; C411703:agouti-related protein-(83-132); D000521:alpha-MSH; D011333:Pro-Opiomelanocortin; C027756:MSH, 4-Nle-7-Phe-alpha-; D000242:Cyclic AMP