Cysteine Residues Are Involved in Structure and Function of Melanocortin 1 Receptor: Substitution of a Cysteine Residue in Transmembrane Segment Two Converts an Agonist to Antagonist

Abstract Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentration of DTT (dithiothreitol) resulted in a decrease in the binding of [ 125 I]-ACTH (adrenocorticotropic hormone, l -isomer) in an uniphasic manner and a decrease in [ 125 I]-NDP-MSH ([Nle 4 ,D-Phe 7 ]-α-melanocyte stimulating hormone; d -isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for α-MSH ( l -isomer) without affecting the affinity of NDP-MSH ( d -isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [ 125 I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists α-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to α-MSH as well as NDP-MSH) generated a cAMP signal in response to α-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at C78G mutant receptor. These findings demonstrate that (i) α-MSH and ACTH ( l -isomers) are different from d -isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by d -isomer agonist (NDP-MSH) but not by l -isomer agonist (α-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at mutant C78G receptor.