The melanocortin-1 receptor carboxyl terminal pentapeptide is essential for MC1R function and expression on the cell surface
Introduction
Mammalian pigmentation depends on the relative amounts and distribution of the different types of melanin pigments in the skin [22]. It has been shown that α-melanocyte-stimulating hormone (α-MSH) acts on human melanocytes to promote differentiation and/or proliferation [1]. This effect is mediated by binding to the melanocortin-1 receptor (MC1R) and activation of the cAMP-signaling pathway [4], ultimately leading to an increase in the total amount of melanin, with a higher ratio of brown to black eumelanins to the reddish pheomelanins. Soon after the cloning of human MC1R [5], [13], it was found that its coding sequence is highly polymorphic, and a number of studies have established the relationship of MC1R haplotype with several characteristics of pigmentation, skin phototype and skin cancer risk (reviewed in [21]).
MC1R belongs to the subfamily of the melanocortin receptors (MCRs), a well-defined subgroup of the G-protein coupled receptors (GPCR) superfamily [5], [11], [21]. The GPCRs display a typical molecular architecture, with an extracellular amino terminus, seven transmembrane (TM) domains and a cytosolic carboxyl terminal region. The length of this C-terminal tail is variable, and within the MCR subfamily it is unusually short, with only 14 amino acids in the human MC1R (Fig. 1), according to current models [15]. Many GPCRs share other structural features, including conserved Cys residues [20]. Some of these Cys residues are located in the extracellular loops of the receptor proteins and may form intramolecular disulfide bridges that stabilize their tertiary structure. This might be the case for MC1R, since mutation to Gly of the extracellular Cys at positions 35, 267, 273 and 275 causes loss of radioligand binding and coupling to the cAMP cascade [6]. Other highly conserved Cys residues are found in the cytosolic tail of many GPCRs [16], [20]. This functionally important domain may be involved in (i) interaction of the ligand–receptor complex with the cognate(s) G-protein(s) [23], (ii) providing signals for correct intracellular traffic and (iii) control of other aspects of receptor function, such as desensitization. Some of these functions are mediated by acylation (palmitoylation or myristoylation) of conserved C-terminal Cys residues and integration of the acyl chain within the lipid bilayer (reviewed in [16], [20]).
In spite of its potentially important role, little attention has been paid to the MC1R C-terminal cytosolic tail. It is generally assumed that the only Cys residue present in this domain, Cys315, is acylated [6], but, as far as we know, a clear demonstration of this possibility has not been presented. Other functional aspects of this region of MC1R have not been analyzed. Interestingly, and in spite of the highly polymorphic nature of the human MC1R gene, no natural point mutations resulting in amino acid changes have been described so far in this region of MC1R. Conversely, a natural R306ter mutation with premature truncation of the C-terminal tail was found in domestic dogs, with homozygous animals showing pheomelanic coats [14]. However, it is not known whether the R306ter mutation results in complete or partial loss-of-function. The molecular basis of the likely functional impairment, as well as the minimal length of the C-terminal tail supporting full receptor function, is also unknown.
We describe here a study of the functional relevance of the human MC1R C-terminus, performed by generation of deleted mutants and by alanine scanning mutagenesis of selected residues. We show that structural integrity of the C- terminus is absolutely required for receptor function, most likely by determining the number of receptor molecules available at the plasma membrane of the cells.
Section snippets
Materials
The non-ionic detergent Igepal CA-630, BSA, EDTA, PMSF and bicinchoninic acid were from Sigma (St. Louis, MO). α-MSH and the superpotent analog [Nle4, d-Phe7] α-MSH (NDP-MSH) were from Calbiochem (Darmstadt, Germany). Reagents and plasticware for cell culture were obtained from either Nunc (Hereford, UK) or Gibco (Paisley, UK). Other reagents were from Merck (Darmstadt, Germany) or Prolabo (Barcelona, Spain) unless specified otherwise.
Expression constructs
All expression constructs were prepared with the pcDNA3
Functional characterization of MC1R variants deleted at the C-terminus
As a first step to study the functional role of the MC1R C-terminal region, we generated two mutants designated Δ14- and Δ12-MC1R. In Δ14-MC1R, the last 14 amino acids were deleted, whereas Δ12-MC1R is equivalent to the dog R306ter variant, with deletion of the C-terminal 12 amino acids (Fig. 1). The constructs were transiently expressed in HEK 293T cells, and analyzed for binding of [125I]NDP-MSH and ability to activate cAMP synthesis in response to agonists. Δ14- and Δ12-MC1R were absolutely
Discussion
In this study, we assessed the importance of the C-terminal cytosolic tail of human MC1R. Using a well characterized heterologous expression system, we found that deletion of the last five amino acids is sufficient to abolish completely MC1R function. Previous genetic studies of different breeds of domestic dogs identified the R306ter variant as a likely loss-of-function allele associated with pheomelanic fur, but did not establish the extent and nature of the functional impairment [14]. The
Conclusions
This study has identified the MC1R C-terminal pentapeptide as a major functional determinant that allows for normal expression of receptor molecules on the plasma membrane. Within this pentapeptide, acylation of Cys315 likely contributes to proper traffic and/or stability of the protein. However, the loss of receptor molecules on the plasma membrane is more pronounced in the Δ5 and Δ3 constructs than in the Cys315Ala mutant. In addition, the Δ1 form and a Thr314Ala mutant also display a reduced
Acknowledgements
Supported by grant SAF2003-03411 from the MCyT and by FEDER. Jesús Sánchez is recipient of a fellowship from the Fundación Séneca, Comunidad Autónoma de la Región de Murcia, Murcia and Berta López Sánchez-Laorden holds a FPI fellowship from the MEC, Spain.
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