The structure of insulin receptor and its subunits. Evidence for multiple nonreduced forms and a 210,000 possible proreceptor.
We have identified the subunits of the insulin receptor using immunoprecipitation by antibodies to the insulin receptor after either biosynthetic or surface labeling of cultured human lymphocytes (IM-9). With this approach, we have found there are two major, Mr = 135,000 (alpha), Mr = 95,000 (beta) and one minor, Mr = 210,000 (gamma) subunit. Peptide mapping clearly demonstrates that the major peptides of the alpha and beta subunits are different, whereas similarities exist in the peptide fragments of the gamma subunit and the alpha and beta subunits after limited proteolysis. The gamma subunit, however, is not simply a disulfide heterodimer of alpha and beta subunits, since this subunit was not reduced by 100 mM dithiothreitol plus 5% 2-mercaptoethanol, or even under more potent denaturing conditions, such as 8 M guanidine-HCL and mercaptoethanol at pH 10.5. In nonreduced gels, free insulin receptor subunits are observed, as well as two higher molecular weight bands of Mr = 520,000 and 350,000. On reduction, the 520,000 band was composed primarily of Mr = 210,000 and 95,000 subunits, whereas the 350,000 band was composed primarily of Mr = 135,000 and 95,000 subunits. These data suggest that the two major subunits of the insulin receptor (alpha and beta) are distinct. In addition, there is a third component of the receptor identifiable of 210,000 which may be a proreceptor or some closely associated effector protein. Furthermore, it appears that in the native state several kinds of disulfide oligomers of these subunits exist. These findings suggest a complex model for insulin receptor synthesis and insertion into the membrane.
D002460:Cell Line; D004229:Dithiothreitol; D006801:Humans; D008214:Lymphocytes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011489:Protein Denaturation; D011498:Protein Precursors / Q000302:isolation & purification; D011972:Receptor, Insulin / Q000302:isolation & purification*; D014357:Trypsin
D010446:Peptide Fragments; D011498:Protein Precursors; D011972:Receptor, Insulin; D014357:Trypsin; D004229:Dithiothreitol