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.
Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-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 alpha-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 [125I]-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) generated a cAMP signal in response to alpha-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 the C78G mutant receptor. These findings demonstrate that (i) alpha-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 (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.
Frändberg
P A
PA
Division of Biological Research on Drug Dependence, Biomedical Centre, Uppsala, S-751 24, Sweden.
Doufexis
M
M
Kapas
S
S
Chhajlani
V
V
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011869:Radioligand Assay; D018043:Receptors, Corticotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044101:Receptors, Melanocortin; D013329:Structure-Activity Relationship; D014162:Transfection; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D004274:DNA, Recombinant; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D003545:Cysteine; D004229:Dithiothreitol