Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Abstract

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent M r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M r 145 000 and an M r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.