title: The melanocortin 4 receptor: oligomer formation, interaction sites and functional significance.
abstract: This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC(4)R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC(4)R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC(4)R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC(4)R.
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Science, University of Leeds, Leeds LS2 9JT, UK. kchapman@imperial.ac.uk
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Chapman
authors.forename: John B C
authors.initials: JB
authors.lastname: Findlay
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D051786:Cyclic AMP Response Element Modulator / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry* / Q000378:metabolism; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; C470072:MC4R protein, human; D044105:Receptor, Melanocortin, Type 4; D051786:Cyclic AMP Response Element Modulator; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D019204:GTP-Binding Proteins