Initial processing of the insulin receptor precursor in vivo and in vitro.
To evaluate the synthesis and initial processing of the insulin receptor precursor, we compared cell-free translation of rat liver poly(A)+ RNA in a reticulocyte lysate system with metabolically labeled rat hepatoma (Fao) cells. In in vitro translation assays, the primary L-[35S]cysteine-labeled products of rat liver mRNA specifically immunoprecipitable with insulin receptor antiserum were two closely migrating polypeptides with a Mr range of 160,000-164,000 (n = 7). This is similar to the size predicted by the insulin receptor cDNA sequence. When heterologous microsomal membranes were included in the cell-free system to process newly synthesized proteins co-translationally, the receptor precursors migrated as larger species of 180 +/- 2 kDa (n = 3). For comparison, when Fao cells were treated with tunicamycin to block core N-glycosylation and pulse-labeled with L-[35S]methionine, two closely migrating precursors were labeled that co-migrated with the unprocessed in vitro translation products (approximately 160 kDa). Pulse labeling of Fao cells in the absence of tunicamycin revealed receptor precursor species of 188 and 198 kDa that rapidly disappeared (t1/2 = 54 min) as the receptor subunits were observed. Thus, the initial products of insulin receptor mRNA translation are two approximately 162-kDa polypeptides that are rapidly processed in intact cells and can only be observed by in vitro studies or by using inhibitors of core glycosylation. Insulin proreceptor species can also be partially glycosylated during cell-free translation by added microsomal membranes. This is the first description of cell-free translation of the insulin proreceptor in a system that will allow detailed characterization of the earliest steps in insulin receptor biogenesis.
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
B J
BJ
Goldstein
C R
CR
Kahn
D000818:Animals; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D011498:Protein Precursors; D014415:Tunicamycin; D011972:Receptor, Insulin