Regulation of Human Melanocortin 1 Receptor Signaling and Trafficking by Thr-308 and Ser-316 and Its Alteration in Variant Alleles Associated with Red Hair and Skin Cancer

Abstract

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/ S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations. The human melanocortin 1 receptor (MC1R)3 is a G proteincoupled receptor preferentially expressed in epidermal melanocytes (1). Within melanocytes, MC1R regulates the amount and type of pigment production and is a major determinant of skin phototype, sensitivity to ultraviolet (UV) radiation-induced damage, and skin cancer risk (2). Interaction with its ligands, ␣ melanocyte-stimulating hormone (␣MSH) or adrenocorticotropin, stimulates cAMP synthesis via the G s protein. cAMP is responsible for most if not all the melanogenic actions of ␣MSH (3). These include activation of tyrosinase, the rate-limiting enzyme in melanin biosynthesis, and a switch from the production of light-colored and poorly photoprotective pheomelanins to synthesis of darker and more photoprotective eumelanins (4). Tyrosinase activation occurs mainly at the transcriptional level and is mediated by induction of microphthalmia, a helix-loophelix transcription factor (5). Melanin synthesis is confined in specialized organelles called melanosomes. The super-potent MC1R agonist norleucine 4 D-phenylalanine-7-melanocyte-stimulating hormone (NDP-MSH) also increases the number and size of melanosomes in mouse melanoma cells (6), s...

Differential and competitive regulation of human melanocortin 1 receptor signaling by β-arrestin isoforms

“…On the other hand, ARRB1 had no inhibitory effect. MC1R internalization was next assessed by an acid-wash method that discriminates external from internal (acid-wash resistant) agonist-receptor complexes (Herraiz et al, 2011b;Sánchez-Laorden et al, 2007). In line with equilibrium binding data, ARRB2 isoforms increased MC1R internalization in the presence of agonist, whereas ARRB1 had no effect (Fig.…”

“…We have previously shown that human MC1R desensitization is mediated by GRK2-or GRK6-dependent phosphorylation of amino acid residues T308 and S316 located in the C-terminal extension, and that MC1R is endocytosed following agonist binding (Sánchez-Laorden et al, 2007;Sánchez-Más et al, 2005a). However, the role of nonvisual ARRB isoforms in regulating MC1R signaling remains unknown.…”

Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with Gαs

“…Samples were centrifuged (105,000 ϫ g, 30 min) and a volume of supernatant containing 10 g of protein was mixed (2:1 ratio) with sample buffer (180 mM Tris-HCl, pH 6.8, 15% glycerol, 9% SDS, 0.075% bromphenol blue, and 7.5% ␤-mercaptoethanol). Electrophoresis and Western blotting were performed as described (28,29). Comparable loading was ascertained by stripping and reprobing the membranes with an anti-ERK2 antibody.…”

“…We next compared agonist-induced internalization. We used an acid wash protocol that distinguishes acid-sensitive radiolabeled agonist bound to the MC1R at the cell surface from acid wash-resistant internalized ligand (29). Only minor differences in the internalization rate were detected for WT or K0-MC1R, in the presence or absence of MGRN1s (Fig.…”

Familial Melanoma in Italy: A Review

“…A functional impairment in MC1R signalling alters the downstream pigmentation pathways, causing an accumulation of pheomelanin that, in turn, has reduced UV protective capacity and can produce cytotoxic and mutagenic metabolites (Sturm, 1998), accelerating melanocytic transformation. This functional impairment can result from different mechanisms: some variants fail in the stimulation of cAMP production (Beaumont et al, 2007;Garcia-Borron et al, 2005), while others show MC1R reduced affinity for its ligand -MSH (Ringholm et al, 2004) or there are even defects in desensitization and internalization of the MC1R receptor itself (Sanchez-Laorden et al, 2007). Given the role of MC1R on epidermal response to UV-induced damage, it is not surprising that the MC1R allelic state influences melanoma risk, by directly regulating skin pigmentation.…”