The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted α-MSH analogues

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of α-MSH, [¹²⁵I-Tyr²,Nle⁴,d-Phe⁷,ATB-Lys¹¹]α-MSH.SDS-PAGE followed by autoradiography showed a single band at 53-56 kDA for the native receptor of 35 kDA after deglycosylated with PNGase F, consistent with the predicted cDNA. Receptor binding studies with α-MSH, γ-MSH and [Nle⁴,d-Phe⁷]α-MSH established that α-MSH and γ-MSH had similar affinities while [Nle⁴,d-Phe⁷]α-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 α-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr² reduced the affinity of the peptide, replacement of Met⁴, Phe⁷, Arg⁸ and Trp⁹ within the peptide core led to a significant loss of affinity. Glu⁵ appeared unimportant for receptor recognition.