In vitro translation of the human insulin proreceptor results in N-linked glycosylation without dimer formation.
The heterotetrameric configuration of the cell surface human insulin receptor (hINSR) is important for mediating insulin action. Investigation of proreceptor dimerization, the quaternary processing event during biogenesis, offers the potential to examine interactions between disulfide-linked receptor subunits. Thus, dimer formation of the proreceptor was examined in a cell-free system that utilized a coupled transcription and translation method with rabbit reticulocyte lysate. Translation products were labeled with [35S]methionine and identified by non-reducing SDS-polyacrylamide gel electrophoresis and autoradiography. In vitro synthesis in the presence of oxidized glutathione failed to demonstrate dimerization of the nascent proreceptor. Co-translational processing with the addition of microsomal membranes resulted in N-linked glycosylation of the proreceptor but without dimer formation. Thus, similar to that observed in vivo, insulin proreceptor dimerization does not appear to be a co-translational or early post-translational event.
Department of Medicine, John A. Burns School of Medicine, University of Hawaii-Manoa.
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Zhou
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Baker
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RF
Arakaki
D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011817:Rabbits; D011972:Receptor, Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D004220:Disulfides; D046911:Macromolecular Substances; D004247:DNA; D008715:Methionine; D011972:Receptor, Insulin; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide