The melanocortin 4 receptor: Oligomer formation, interaction sites and functional significance

• Published in: Biochimica et biophysica acta Vol. 1828; no. 2; pp. 535 - 542
• Main Authors: Chapman, Kathryn L., Findlay, John B.C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-02-2013
• Subjects: Allosteric Site; alpha-MSH - analogs & derivatives; alpha-MSH - pharmacology; Amino Acid Sequence; Binding Sites; Cell Membrane - metabolism; Cross-Linking Reagents - chemistry; Cyclic AMP Response Element Modulator - metabolism; Dimerization; Disulfides - chemistry; Dose-Response Relationship, Drug; GPCR; GTP-Binding Proteins - chemistry; HEK293 Cells; Humans; Ligands; Melanocortin 4 receptor; Mutagenesis; Mutation; Oligomerization; Protein Binding; Receptor, Melanocortin, Type 4 - chemistry; Receptor, Melanocortin, Type 4 - metabolism; Trans-activation; NDP-MSH; Oligomerization; Gpp(NH)p; ACTH; Trans-activation; Gα; CRE-Luc cells; ECL; MC4R; POMC; GTPγS; Melanocortin 4 receptor; HA epitope; G-protein; GPCR; TM; AgRP; CuP
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2012.10.011

This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC4R) 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 MC4R 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 MC4R 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 MC4R.
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► The MC4R is an oligomer. ► C84 located on TMII can crosslink between dimers when coupled to the G-protein. ► The MC4R retains high affinity for the agonist in the absence of the G-protein. ► Agonist occupancy increases in the absence of G-protein. ► Transactivation studies on the MC4R demonstrated crosstalk in the oligomer.