Bioactive Motifs of Agouti Signal Protein Victoria M. Virador,* Chie Santis,* Minao Furumura,* Hubert Kalbacher,† and Vincent J. Hearing* ,1 *Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and †Medical and Natural Sciences Research Center, University of Tu ¨ bingen, D-72074 Tu ¨ bingen, Germany The switch between the synthesis of eu- and pheomelanins is modulated by the interaction of two paracrine signaling molecules, -melanocyte stimulat- ing hormone (MSH) and agouti signal protein (ASP), which interact with melanocytes via the MSH receptor (MC1R). Comparison of the primary sequence of ASP with the known MSH pharmacophore provides no sug- gestion about the putative bioactive domain(s) of ASP. To identify such bioactive motif(s), we synthesized 15- mer peptides that spanned the primary sequence of ASP and determined their effects on the melanogenic activities of murine melanocytes. Northern and West- ern blotting were used, together with chemical analy- sis of melanins and enzymatic assays, to identify three distinct bioactive regions of ASP that down-regulate eumelanogenesis. The decrease in eumelanin produc- tion was mediated by down-regulation of mRNA levels for tyrosinase and other melanogenic enzymes, as oc- curs in vivo, and these effects were comparable to those elicited by intact recombinant ASP. Shorter pep- tides in those motifs were synthesized and their ef- fects on melanogenesis were further investigated. The amino acid arginine, which is present in the MSH pep- tide pharmacophore (HFRW), is also in the most active domain of ASP (KVARP). Our data suggest that lysines and an arginine (in motifs such as KxxxxKxxR or KxxRxxxxK) are important for the bioactivity of ASP. Identification of the specific ASP epitope that inter- acts with the MC1R has potential pharmacological ap- plications in treating dysfunctions of skin pigmenta- tion. Key Words: agouti; melanocortin; receptor; G pro- tein; antagonist. INTRODUCTION G-protein coupled receptors are integral membrane proteins that contain seven transmembrane domains and that mediate cellular responses to a wide array of signaling molecules. In some G-protein coupled recep- tors, one ligand can activate different receptors within the same family, one example being the melanocortin family of receptors which are differentially activated by products of the POMC gene. The melanocortins are a group of melanotropic and adrenocorticotropic hor- mones produced mainly in the pituitary gland, but also in a number of extrapituitary sites, including the skin [1]. The effects of these peptides (known collectively as melanotropins) in different tissues are mediated by a family of specifically expressed melanocortin receptors (MCxR), 2 termed MC1R, MC2R, etc. [2, 3]. The intra- cellular action of melanocortin receptors, as well as other G-protein coupled receptors, is associated with the activation of adenylyl cyclase and the subsequent elevation of intracellular cAMP levels. In melanocytes, this pathway activates the expression of tyrosinase and other melanogenic genes [4, 5]. Mitogen activated pro- tein kinases and various transcription factors are also part of the complex response pathway [6 – 8]. MC1R is primarily expressed by melanocytes and regulates their proliferation and melanogenesis, but it may also be expressed by monocytes, keratinocytes, fibroblasts, and endothelial cells [9 –12]. In wild-type agouti mice, coat color is characterized by a yellow stripe on a black background in each hair as a conse- quence of a temporal (3-day) switch in the production of eu- to pheomelanin and then back again. Two interact- ing factors are known to be involved in regulating that melanogenic switch via the MC1R, namely agouti sig- nal protein (ASP) and -melanocyte stimulating hor- mone (MSH). MSH is a relatively short oligopeptide (SYSMEHFRWGKPV) and, in human skin, it may play important roles in regulating general epidermal cell function in addition to regulating pigmentation [13]. ASP is a much larger, 131 amino acid residue protein with several distinct sequence motifs (Fig. 1) that is expressed in skin. ASP interacts with at least four of the five known melanocortin receptors, and its best characterized interactions are with MC1R and MC4R 1 To whom correspondence and reprint requests should be ad- dressed at Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 1B25, Bethesda, MD 20892. Fax: (301) 402-8787. E-mail: hearingv@nih.gov. 2 Abbreviations used: AGRP, agouti related protein; ASIP, human agouti signal protein; ASP, murine agouti signal protein; Dct, dopachrome tautomerase/TRP2; IGF, insulin-like growth factor; MCxR, melanocortin receptor; MSH, -melanocyte stimulating hor- mone; Tyrp1, tyrosinase related protein 1/TRP1. 54 0014-4827/00 $35.00 Experimental Cell Research 259, 54 – 63 (2000) doi:10.1006/excr.2000.4975, available online at http://www.idealibrary.com on