The receptor for alpha-melanotropin of mouse and human melanoma cells. Application of a potent alpha-melanotropin photoaffinity label.
The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.
Solca
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F
Department of Research (ZLF), University Hospital, Basel, Switzerland.
Siegrist
W
W
Drozdz
R
R
Girard
J
J
Eberle
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D000345:Affinity Labels; D000818:Animals; D002460:Cell Line; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D008545:Melanoma / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009439:Neuraminidase; D010777:Photochemistry; D051381:Rats; D011979:Receptors, Pituitary Hormone / Q000032:analysis* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured / Q000032:analysis / Q000378:metabolism*; D000521:alpha-MSH / Q000378:metabolism*
D000345:Affinity Labels; D006023:Glycoproteins; D011979:Receptors, Pituitary Hormone; D000521:alpha-MSH; C029446:MSH receptor; D009439:Neuraminidase