THE MELANOCORTIN (MC3) RECEPTOR FROM RAT HYPOTHALAMUS - PHOTOAFFINITY-LABELING AND BINDING OF ALANINE-SUBSTITUTED ALPHA-MSH ANALOGS

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a r adiolabelled photoreactive analogue of alpha-MSH, -125-Tyr(2),Nle(4),D -Phe(7),ATB-Lys(11)]alpha-MSH. SDS-PAGE followed by autoradiography sh owed a single band at 53-56 kDa for the native receptor or 35 kDa afte r deglycosylated with PNGase F, consistent with the predicted cDNA seq uence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle(4), D-Phe(7)]alpha-MSH established that alpha-MSH and gamma-MSH had simila r affinities while [Nle(4),D-Phe(7)]alpha-MSH bound 100 times more str ongly. These results suggest that the receptor recognises the conserve d 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides . The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in lig and-receptor interactions. While in the terminal regions only the repl acement of Tyr(2) reduced the affinity of the peptide, replacement of Met(4), Phe(7), Arg(8) and Trp(9) within the peptide core led to a sig nificant loss of affinity. Glu(5) appeared unimportant for receptor re cognition.