title: Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand.
abstract: Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an endogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the availability of an ASIP/agouti radioligand. In this report we describe the functional characteristics of a chemically synthesized truncated ASIP variant, ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radioligand, (125)I-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASIP/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha-melanocyte-stimulating hormone cAMP generation at the cloned human melanocortin receptor (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of inhibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only exhibited weak inhibitory activity at the hMC2R. In competition binding assays with the radioligand (125)I-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] displayed a hierarchy of binding affinity that roughly paralleled its rank order of inhibitory potency at the various MCR subtypes, i.e., hMC1R approximately hMC4R > hMC3R approximately hMC5R > hMC2R. Structure-activity studies revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological potency than either the further truncated ASIP variants ASIP-(116-132) or cyclo(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhibits melanocortin action by directly binding to target MCRs and provide additional insight into the structural requirements for maximal inhibitory potency.
authors.affiliation: Department of Surgery, University of Michigan Medical Center, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0682, USA.
authors.forename: Y K
authors.initials: YK
authors.lastname: Yang
authors.forename: C
authors.initials: C
authors.lastname: Dickinson
authors.forename: Y M
authors.initials: YM
authors.lastname: Lai
authors.forename: J Y
authors.initials: JY
authors.lastname: Li
authors.forename: I
authors.initials: I
authors.lastname: Gantz
mesh_terms: D054366:Agouti Signaling Protein; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009074:Melanocyte-Stimulating Hormones / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*; D011979:Receptors, Pituitary Hormone / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D000521:alpha-MSH / Q000494:pharmacology
chemical_list: D054366:Agouti Signaling Protein; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011506:Proteins; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins; D000521:alpha-MSH; C029446:MSH receptor; D009074:Melanocyte-Stimulating Hormones; D000242:Cyclic AMP