Cysteine residues are involved in structure and function of melanocortin 1receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentration of DTT (dithiothreitol) resulted in a decrease in the binding of [I-125]-ACTH (adrenocorticotropic hormone, L-isomer) in an u niphasic manner and a decrease in [I-125]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha -melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pr etreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity f or alpha -MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isome r). To characterize the role of individual cysteine residues, we employed s ite-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand b inding 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 [I-125]-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 alpha -MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha -MSH as well as NDP-MSH) gen erated a cAMP signal in response to alpha -MSH (identical to wild-type hMC1 R) but interestingly could not be stimulated by NDP-MSH. Moreover, this sin gle amino acid substitution converted NDP-MSH from being an agonist to anta gonist at C78G mutant receptor. These findings demonstrate that (i) alpha - MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensi tivity to DTT for receptor binding, (ii) cysteine residues in N-terminus an d extracellular loop three make disulfide bridges and are needed for struct ural 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 re sponse by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha -MS H), and (v) wild-type receptor agonist NDP-MSH is an antagonist at mutant C 78G receptor. (C) 2001 Academic Press.