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 radiolabelled photoreactive analogue of alpha-MSH, [I-125-Tyr(2),Nle(4),D-Phe(7),ATB-Lys(11)]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle(4),D-Phe(7)]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle(4),D-Phe(7)]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved '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 ligand-receptor interactions. While in the terminal regions only the replacement 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 significant loss of affinity. Glu(5) appeared unimportant for receptor recognition.