alpha-Melanocyte stimulating hormone (alpha-MSH) and ACTH increase the proliferation and melanogenesis of cultured human melanocytes. To further analyze how melanotropins produce these biological effects, we investigated the regulation of the melanocortin receptor MC1R expression by alpha-MSH and ACTH using Northern blot analysis and determine the relative affinity of the receptor for the structurally similar peptides alpha-MSH, ACTH, beta-MSH, and gamma-MSH. We also determined the relative potencies of these hormones to stimulate cAMP formation, tyrosinase activity, and melanocyte proliferation. The order of affinity and potency of the noted melanotropins in these assays were alpha-MSH = ACTH> beta-MSH > gamma-MSH. Because the binding affinity of each of these melanotropins for the MC1R correlated with its ability to stimulate human melanocyte proliferation and melanogenesis, we conclude that these effects are mediated specifically by binding to and activation of the MC1R. gamma-MSH stimulated cAMP formation without affecting proliferation or melanogenesis. However, we found that relative to alpha-MSH, the effect of gamma-MSH on cAMP formation was transient. Our results suggest that alpha-MSH, ACTH, and possibly beta-MSH, but not gamma-MSH, are capable of a physiological role in regulating human pigmentation, and that melanocytes in human skin are a specific target for these hormones.

In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

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the implications of these variants on the function of the The cloning and characterization of the human melanocortin-1 receptor (MC1R) and the demonstration that normal human MC1R revealed the following. Human melanocytes homelanocytes respond to the melanocortins, a-melanocyte stimmozygous for Arg160Trp mutation in the MC1R demonulating hormone (a-MSH) and adrenocorticotrophic hormone strated a significantly reduced response to a-MSH. Also, this culture responded poorly to ASP and exhibited an exagger-(ACTH), with increased proliferation and eumelanogenesis ated cytotoxic response to UVR. Another culture, which was had put an end to a long-standing controversy about the role homozygous for Val92Met mutation in the MC1R, demonof melanocortins in regulating human cutaneous pigmentation. strated a normal response to a-MSH. Heterozygous mutaWe have shown that a-MSH and ACTH bind the human tions that are frequently expressed in various melanocyte MC1R with equal affinity, and are equipotent in their mitocultures did not disrupt MC1R function. These results begin genic and melanogenic effects on human melanocytes. We also to elucidate the significance of MC1R variants in the function showed that the activation of the MC1R is important for the of the receptor. Our data emphasize the significance of a melanogenic response of human melanocytes to ultraviolet normally functioning MC1R in the response of melanocytes to radiation (UVR). The MC1R is also the principal mediator of melanocortins, ASP, and UVR. the inhibitory effects of agouti signaling protein (ASP) on melanogenesis. Expression of the MC1R is subject to regulation by its own ligands a-MSH and ACTH, as well as by Key words: Melanocortin 1 receptor, Human melanocytes, Ultraviolet radiation, Melanocortins, Agouti signaling protein, UVR and endothelin-1. Recent studies that we conducted on the expression of MC1R variants by human melanocytes and Eumelanin, Pheomelanin that have an extensive amino acid sequence homology. These peptides include a-melanocyte stimulating hormone (a-MSH), b-MSH, g-MSH, and adrenocorticotrophic hormone (ACTH), all of which are derived from a large precursor peptide, pro-opiomelanocortin (POMC) (1). The 4-10 carboxy teminus amino acid residues of a-MSH, b-MSH,