ANTAGONIST AND AGONIST ACTIVITIES OF THE MOUSE AGOUTI PROTEIN FRAGMENT (91–131) AT THE MELANOCORTIN-1 RECEPTOR

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Abstract
Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-α-MSH (Ki 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP91–131; the IC50 values for AP91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91–131 with IC50 values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91–131 in the adenylate cyclase and melanin assays. On the other hand, AP91–131 inhibited cell growth similar to α-MSH (IC50 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.