The melanocytehyphen;stimulating hormone lpar;MSHrpar; receptor in M2R mouse melanoma tumourssolubilization and properties of the receptorhyphen;MSH complex and its covalently crosslinked conjugate

摘要

Several properties of the MSH receptor in solid melanotic and amelanotic mouse M2R tumour Isografts were studied in C57BL mice. Using cell membrane fractions prepared from such tumours and the superpotent Nle4.D-Phe7alpha;MSH analogue, the affinity and receptor contents of the two 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 30percnt; within 12 days at 4deg;C. While this high stability can be maintained in the pH range of 7.0ndash;8.5, the solubllized R-MSH complex becomes Increasingly unstable below pH 7.0 and totally dissociates at a pH 6.0. In the membrane-bound form, the R. MSH complex shows a parallel pH stability profile which is shifted down by approximately two pH units. In addition to low pH, the R-MSH complex becomes unstable and totally dissociates in the presence of 10 mM EGTA, suggesting that the calclum-sensitive function of the receptor is still associated with the receptor in the detergent-soluble state. The R-MSH complexes in the soluble and membrane-bound forms are also totally resistant to proteolytic digestion by V8 protease, but were slowly digested by trypsin. Treatment of R-MSH with 1-ethyl-3-)3-dimethylemlno-propyl)carbodllmide hydrochloride or bis (sulphosuccinimidyl) suberate led to covalent crosslinking of MSH to the receptor molecule. The electrophoretic mobility on SOS-PAGE of the 43/46 kD doublet of the receptor-MSH conjugate (Rast;MSH) was Identical to we photoaffinity labelled MSH receptor product described earlier in cultured M2R cells. However, the efficiency of production of the crosslinked product was 30percnt;, much higher than that achieved previously by photoaffinity labelling. Using rabbit porycional anti-alpha;MSH antibodies, the Rast;MSH conjugate was Identifiable on Western Immunobiots) These results provide a basis for further development of procedures for purification of the MSH receptor molecule and studying Ms protein structure.