Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.
In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
J E
JE
Gerst
J
J
Sole
E
E
Hazum
Y
Y
Salomon
D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D009074:Melanocyte-Stimulating Hormones / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D011979:Receptors, Pituitary Hormone / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D011979:Receptors, Pituitary Hormone; C057204:azidoiodo-beta-MSH; C029446:MSH receptor; D009074:Melanocyte-Stimulating Hormones