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.

Molecular Determination of Agouti-Related Protein Binding to Human Melanocortin-4 Receptor

“…We have previously shown that the chemically synthesized C-terminal region of AGRP com- petitively antagonizes ␣-MSH at melanocortin receptors with potency equal to that of the full-length proteins (Yang et al, 1999a). This is consistent with similar findings for agouti (Yang et al, 2001). In this study, we examine the molecular basis of ligand-receptor interaction between AGRP and hMC4R by pharmacologically studying AGRP fragments and analyzing the function of melanocortin chimeric receptors.…”

Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling

“…This cell line has previously been characterized [36, 37]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) with 100 U/ml penicillin and 100 U/ml streptomycin.…”

Structures of the Agouti Signaling Protein

“…As shown in Figure 2 The Structures of ASIP exhibits a slope of near unity, demonstrating competitive antagonism, and K i values, determined from the intercept, lie within the range previously established for both full-length and C-terminal ASIP ( Table 2). [17][18][19] NMR structure of ASIP-YY Structure determination was performed using the same strategy previously applied to AgRP(87-132). 11 Specifically, a standard suite of homonuclear Table 2 and insets.)…”