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