Mutational analysis of melanocortin receptors 1 and 5

Sammanfattning: The melanocortin receptor family includes five subtypes, named MC1R to MC5R. Melanocortin receptors are predominantly coupled to second messenger pathway of cyclic AMP through coupling to G-protein. This study is focused on structure-function relationship of MC1R and MC5R. Results demonstrate that (i) mutations D117A and H260A in MC1R decrease the affinity for natural melanocortins (L-isomer) but not for the synthetic analogue NDP-MSH (D-isomer), (ii) reduction of disulphide bonds in MC1R with DTT resulted in decrease in binding of 125I-ACTH in an uniphasic manner and decrease in binding of 125I-NDP-MSH in biphasic manner (iii) single point mutations of four cysteine residues to glycine in extracellular loops of MC1R resulted in a complete loss of ligand binding, (iv) mutant at position C78 in MC1R with wild type ligand binding generated a cAMP signal only in response to α-MSH but not to NDP-MSH, moreover,this single amino acid substitution converted NDP-MSH from being an agonist at wild type receptor to antagonist at this mutant receptor (v) several residues in third intracellular loop of MC1R were identified as necessary for generating a functional response, and a sequence of four amino acids K226-R227-Q228-R229 was found to be essential for coupling of MC1R to G-protein, (vi) a human MC1R variant with a mutation at codon 151 (R151C), cloned from genomic DNA obtained from an individual with red hair and fair skin was found to exhibit wild-type affinities to melanocortins, but was completely nonfunctional, in terms of generating an agonist induced cAMP response (vii) two positions in humanMC5R, Q235 and R272, conserved in all other melanocortin receptors as lysine and cysteine, was demonstrated to raise melanocortin affinities for mutant Q235K and R272C compared to wild-type human MC1R. Thus, this study has identified amino acid residues contributing to ligand interaction and functional activation of melanocortin receptors.