Antagonist and agonist activities of the mouse Agouti protein fragment (91-131) at the melanocortin-1 receptor

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP(91-131)) 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 alpha -melanocyte-stimulating hormone (alpha -MSH) in displacing the radioligand [I-125]-[Nle(4), D-Phe(7)]-alpha -MSH (K-i 6.5 +/-0.8 nmol/l). alpha -MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP(91-131); the IC50 values for AP(91-131) 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 AP(91-131) with IC50 values of 9.6 +/-1.8 nM and 5.0 +/-2.4 nM, respectively. This indicates inverse agonist activity of AP(91-131) Similar to that of native AR 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 alpha -MSH (IC50 11.0 +/-2.1 nM; maximal inhibition 1.8-fold higher than that of alpha -MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha -MSH. These results demonstrate that AP(91-131) 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 alpha -MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.