Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand

Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an end ogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the av ailability of an ASIP/agouti radioligand. In this report we describe the fu nctional characteristics of a chemically synthesized truncated ASIP variant , ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radi oligand, I-125-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASI P/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha -melanocyte- stimulating hormone cAMP generation at the cloned human melanocortin recept or (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of in hibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only e xhibited weak inhibitory activity at the hMC2R. In competition binding assa ys with the radioligand I-125-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] di splayed a hierarchy of binding affinity that roughly paralleled its rank or der of inhibitory potency at the various MCR subtypes, i.e., hMC1R approxim ate to hMC4R > hMC3R approximate to hMC5R > hMC2R. Structure-activity studi es revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological pot ency than either the further truncated ASIP variants ASIP-(116-132) or cycl o(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhi bits melanocortin action by directly binding to target MCRs and provide add itional insight into the structural requirements for maximal inhibitory pot ency.