Mechanism of dimerization of the human melanocortin 1 receptor

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Abstract

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.

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Materials and methods

Materials. Igepal CA-630, BSA, EDTA, PMSF, iodoacetamide and bicinchoninic acid were from Sigma (St. Louis, MO). The synthetic αMSH analogue [Nle4, D-Phe7] αMSH (NDP-MSH) was from Calbiochem (Darmstadt, Germany). The radioligand [125I]NDP-MSH, specific activity 2000 Ci/mmol, and a cAMP radioimmunoassay kit were from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Other reagents were from Merck (Darmstadt, Germany) or Prolabo (Barcelona, Spain).

Expression constructs and

Results and discussion

Dimerization of the MC1R was previously demonstrated by co-immunoprecipitation studies [17]. The mechanisms of GPCR dimerization are often incompletely understood, but may involve: (i) coiled-coil interactions between the C-terminal domains, (ii) covalent intersubunit disulfide bonding, and (iii) the swapping of large receptor domains to yield functional units that comprise structural elements from each partner. We have analyzed the contribution of these three types of intersubunit interactions

Acknowledgments

Supported by Grant SAF2006-11206 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain, and FEDER funds (European Community). BLS-L and ABP-O hold fellowships from the Ministry of Education (Spain), and CH from the Fundación Séneca (CARM). PTZ was a UNESCO-L’OREAL fellow for Women in Science in 2005 and a fellow from the Puglia Region (Italy). We thank Prof. R. Peñafiel and A. López-Contreras (Department of Biochemistry and Molecular Biology, University of Murcia) for gift

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