THE MELANOCYTE-STIMULATING HORMONE (MSH) RECEPTOR IN M2R MOUSE MELANOMA TUMORS - SOLUBILIZATION AND PROPERTIES OF THE RECEPTOR MSH COMPLEX AND ITS COVALENTLY CROSS-LINKED CONJUGATE

Several properties of the MSH receptor in solid melanotic and amelanot ic mouse M2R tumour isografts were studied in C57BL mice. Using cell m embrane fractions prepared from such tumours and the superpotent [Nle4 ,D-Phe7]alphaMSH analogue, the affinity and receptor contents of the t wo tumour variants were found to be similar. When occupied by MSH, the receptor-MSH complex (R . MSH) was readily soluble in cholate. In the solubilized form, R . MSH was extremely stable and dissociated to an extent of only 30% within 12 days at 4-degrees-C. While this high stab ility can be maintained in the pH range of 7.0-8.5, the solubilized R . MSH complex becomes increasingly unstable below pH 7.0 and totally d issociates at a pH < 6.0. In the membrane-bound form, the R . MSH comp lex shows a parallel pH stability profile which is shifted down by app roximately two pH units. In addition to low pH, the R . MSH complex be comes unstable and totally dissociates in the presence of 10 mM EGTA, suggesting that the calcium-sensitive function of the receptor is stil l associated with the receptor in the detergent-soluble state. The R . MSH complexes in the soluble and membrane-bound forms are also totall y resistant to proteolytic digestion by V8 protease, but were slowly d igested by trypsin. Treatment of R . MSH with 1-ethyl-3-(3-dimethylami no-propyl)carbodiimide hydrochloride or bis (sulphosuccinimidyl) suber ate led to covalent crosslinking of MSH to the receptor molecule. The electrophoretic mobility on SDS-PAGE of the 43/46 kD doublet of the re ceptor-MSH conjugate (RMSH) was identical to the photoaffinity labell ed MSH receptor product described earlier in cultured M2R cells. Howev er, the efficiency of production of the crosslinked product was approx imately 30%, much higher than that achieved previously by photoaffinit y labelling. Using rabbit polyclonal anti-alphaMSH antibodies, the RM SH conjugate was identifiable on Western immunoblots. These results pr ovide a basis for further development of procedures for purification o f the MSH receptor molecule and studying its protein structure.