The melanocyte-stimulating hormone (MSH) receptor in M2R mouse melanoma tumours: solubilization and properties of the receptor-MSH complex and its covalently crosslinked conjugate.
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-Phe7]alpha 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 30% within 12 days at 4 degrees C. While this high stability 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 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 calcium-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-dimethylamino-propyl)carbodiimide hydrochloride or bis (sulphosuccinimidyl) suberate led to covalent crosslinking of MSH to the receptor molecule. The electrophoretic mobility on SDS-PAGE of the 43/46 kD doublet of the receptor-MSH conjugate (R*MSH) was identical to the photoaffinity labelled MSH receptor product described earlier in cultured M2R cells. However, the efficiency of production of the crosslinked product was approximately 30%, much higher than that achieved previously by photoaffinity labelling. Using rabbit polyclonal anti-alpha MSH antibodies, the R*MSH conjugate was identifiable on Western immunoblots. These results provide a basis for further development of procedures for purification of the MSH receptor molecule and studying its protein structure.
Shafir
I
I
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Schmidt-Sole
J
J
Shai
E
E
Salomon
Y
Y
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents; D004355:Drug Stability; D007457:Iodine Radioisotopes; D009074:Melanocyte-Stimulating Hormones / Q000276:immunology / Q000378:metabolism*; D018328:Melanoma, Amelanotic / Q000378:metabolism* / Q000648:ultrastructure; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D011817:Rabbits; D011979:Receptors, Pituitary Hormone / Q000032:analysis / Q000378:metabolism*; D012878:Skin Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D012995:Solubility; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000378:metabolism
D000906:Antibodies; D003432:Cross-Linking Reagents; D003902:Detergents; D007457:Iodine Radioisotopes; D011979:Receptors, Pituitary Hormone; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; C029446:MSH receptor; D009074:Melanocyte-Stimulating Hormones