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Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of a-MSH, ['251-Tyr2,Nle4,0-Phe7,ATB-Lys"]a-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 a-MSH, y-MSH and [Nle4,0-Phe'Ja-MSH established that a-MSH and y-MSH had similar affinities while [Nle4,D-Phe7]a-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 a-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr' reduced the affinity of the peptide, replacement of Met4, Phe', Arg* and Trp' within the peptide core led to a significant loss of affinity. Glu' appeared unimportant for receptor recognition.

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