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

The melanocortin 1 receptor (MC1R) is a G-protein-coupled receptor (GPCR) crucial for the regulation of melanocyte proliferation and differentiation. MC1R activation by melanocortin hormones triggers the cAMP pathway and stimulates the extracellular-signal-regulated protein kinases ERK1 and ERK2 to promote synthesis of photoprotective eumelanin pigments, among other effects. Signaling from most GPCRs is regulated by the ß-arrestin (ARRB) family of cytosolic multifunctional adaptor proteins, which mediate signal termination and endocytosis of GPCR-agonist complexes. The ubiquitously expressed non-visual ß-arrestin1 (ARRB1) and ß-arrestin2 (ARRB2) are highly similar but not functionally equivalent. Their role in the regulation of MC1R is unknown. Using a combination of co-immunoprecipitation, gel filtration chromatography, confocal microscopy, siRNA-mediated knockdown and functional assays, we demonstrated agonist-independent competitive interactions of ARRB1 and ARRB2 with MC1R, which might also be independent of phosphorylation of Ser/Thr residues in the C-terminus of the MC1R. The effects of ARRBs were isoform specific; ARRB2 inhibited MC1R agonist-dependent cAMP production but not ERK activation, stimulated internalization and showed prolonged co-localization with the receptor in endocytic vesicles. By contrast, ARRB1 had no effect on internalization or functional coupling, but competed with ARRB2 for binding MC1R, which might increase signaling by displacement of inhibitory ARRB2. These data suggest a new mechanism of MC1R functional regulation based on the relative expression of ARRB isoforms, with possible activatory ARRB1-dependent effects arising from partial relief of inhibitory ARRB2-MC1R interactions. Thus, competitive displacement of inhibitory ARRBs by functionally neutral ARRB isoforms might exert a paradigm-shifting signal-promoting effect to fine-tune signaling downstream of certain GPCRs.

N-glycosylation of the human melanocortin 1 receptor: occupancy of glycosylation sequons and functional role.

The melanocortin 1 receptor (MC1R), a major determinant of skin pigmentation and phototype, mediates the actions of α-melanocyte-stimulating hormone on melanocytes and is critical for melanocyte proliferation and differentiation. MC1R has two putative N-glycosylation targets, Asn15 and Asn29. It has been shown that MC1R is a glycoprotein with an unusual sensitivity to endoglycosidase H digestion. However, the occupancy and functional importance of each specific glycosylation sequon remains unknown. We demonstrate that MC1R is N-glycosylated at Asn15 and Asn29, with structurally and functionally different glycan chains. N-glycosylation is not necessary for high affinity agonist binding or functional coupling but has a strong effect on the availability of MC1R molecules on the plasma membrane, most likely by a combination of improved forward trafficking and decreased internalization. Finally, we found that MC1R variants exhibit different degrees of glycosylation which do not show a simple correlation with their functional status or intracellular trafficking.

Subcellular localization of antizyme inhibitor 2 in mammalian cells: Influence of intrinsic sequences and interaction with antizymes.

Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme-binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER-Golgi intermediate compartment (ERGIC) and in the cis-Golgi network, apparently not related to any known cell-sorting sequence. Our results rather suggest that the N-terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis.

Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady-state retention of mutant forms in the proximal golgi.

The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to reddish, poorly photoprotective pheomelanins. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation (the RHC phenotype) and increased skin cancer risk. Three highly penetrant RHC variants, R151C, R160W, and D294H are loss-of-function MC1R mutants with altered cell surface expression. In this study, we show that forward trafficking was normal for D294H. Conversely, export traffic was impaired for R151C, which accumulated in the endoplasmic reticulum (ER), and for R160W, which was enriched in the cis-Golgi. This is the first report of steady-state retention in a post-ER secretory compartment of a GPCR mutant found in the human population. Residues R151 and R160 are located in the MC1R second intracellular loop (il2). Two other mutations in il2, T157A preventing T157 phosphorylation and R162P disrupting a (160)RARR(163) motif, also caused intracellular retention. Moreover, T157 was phosphorylated in wild-type MC1R and a T157D mutation mimicking constitutive phosphorylation allowed normal traffic, and rescued the retention phenotype of R160W and R162P. Therefore, MC1R export is likely regulated by T157 phosphorylation and the (160)RARR(163) arginine-based motif functions as an ER retrieval signal. These elements are conserved in mammalian MC1Rs and in all five types of human melanocortin receptors. Thus, members of this GPCR subfamily might share common mechanisms for regulation of plasma membrane expression.

Mechanism of dimerization of the human melanocortin 1 receptor.

The melanocortin 1 receptor (MC1R) is a dimeric G protein-coupled receptor expressed in melanocytes, where it regulates the amount and type of melanins produced and determines the tanning response to ultraviolet radiation. We have studied the mechanisms of MC1R dimerization. Normal dimerization of a deleted mutant lacking the seventh transmembrane fragment and the C-terminal cytosolic extension excluded coiled-coil interactions as the basis of dimerization. Conversely, the electrophoretic pattern of wild type receptor and several Cys-->Ala mutants showed that four disulfide bonds are established between the monomers. Disruption of any of these bonds abolished MC1R function, but only the one involving Cys35 was essential for traffic to the plasma membrane. A quadruple Cys35-267-273-275Ala mutant migrating as a monomer in SDS-PAGE in the absence of reducing agents was able to dimerize with WT, suggesting that in addition to disulfide bond formation, dimerization involves non-covalent interactions, likely of domain swap type.

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.

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.

Dimerization of the human melanocortin 1 receptor: functional consequences and dominant-negative effects.

The melanocortin 1 receptor (MC1R), a G(S)-protein-coupled receptor (GPCR), is a key regulator of proliferation and differentiation of epidermal melanocytes, and a determinant of human skin phototype and cancer risk. Homodimerization has been demonstrated for several GPCRs, but little information is available for MC1R. SDS-PAGE analysis of melanoma cells and heterologous cells expressing epitope-tagged MC1R revealed dimeric and oligomeric species in detergent-solubilized extracts, confirmed by co-immunoprecipitation of differentially tagged MC1R forms. Dimerization occurs early during MC1R biosynthesis, and is seen for mutants displaying intracellular retention. These mutants exerted dominant-negative effects on wild-type (WT) MC1R. Conversely, partial functional trans-complementation of selected loss-of-function mutants was observed. WT-MC1R lacks cooperativity in agonist binding, yet coexpression of WT and a C-terminal deletion mutant yielded a form of different pharmacological properties. The natural diminished function alleles R151C, R160W, and D294H, associated with red hair, displayed dimerization and heterodimerization with WT. Coexpression of WT and R151C or R160W reduced the density of binding sites on the plasma membrane of transfected cells, whereas D294H mediated a dominant-negative effect on functional coupling to adenylyl cyclase. Therefore, subtle changes of functional properties may be associated with different MC1R haplotypes, contributing to the complexity of skin phenotype.

Melanocortin-1 receptor structure and functional regulation.

The melanogenic actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R). MC1R is a member of the G-protein-coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or alpha-melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist-independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.