M Doufexis | PubMed Health

PubMed2001

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.

Adrenocorticotropic Hormone↗

PubMed1998

Human pigmentation phenotype: a point mutation generates nonfunctional MSH receptor.

alpha-Melanocyte stimulating hormone (alpha-MSH) regulates skin and hair pigmentation by modulating the activity of MSH receptor (MC1R). We have identified Arg151Cys variant of human MC1R in genomic DNA isolated from a person with red hair and light skin of type I. The Arg151Cys variant of MC1R binds to radio-labelled analogue of alpha-MSH with identical affinity as wild type MC1R but can not be stimulated to produce cyclic AMP (cAMP). The mutation Arg151Cys renders human MC1R completely nonfunctional, which explains the red hair, light skin and poor tanning ability (skin type I). This is the first report ever describing a nonfunctional MC1R isolated from a human subject.

Animals↗

PubMed1998

Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling.

To delineate domains essential for G-protein coupling in melanocortin 1 receptor (MC1R), we mutated polar and basic residues to alanine at eleven positions in the putative third intracellular loop and determined consequent changes in the ligand binding and generation of second messenger cAMP. Results demonstrate that ligand binding affinity was not affected by any of the mutations. However, every mutant displayed reduced functional response as compared to the wild type receptor. Replacement of residues (K226, R227, Q228, R229, H232, Q233 and K238) present in second half of third intracellular loop resulted in an almost complete loss of functional response. The results have demonstrated that the amino acid residues present in C-terminal portion of third intracellular loop of MC1R are involved in coupling to G-protein and that a region of four amino acids, K226-R227-Q228-R229 is essential for coupling of MC1R to G-protein.

Amino Acid Sequence↗