Role of glycosylation in the processing of newly translated insulin processing of newly translated insulin proreceptor in 3T3-L1 adipocytes

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [³⁵S]methionine after which ³⁵S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first ³⁵S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t( 1/2 ) ≃ 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t( 1/2 ) ≃ 2 h) proteolytically processed to 125-kDa (α') and 83-kDa (β') precursors of the mature α- and β-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the α'- and β'-precursor polypeptides were converted to the mature 135-kDa α- and 95-kDa β-receptor sub-units. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa α'- and 83-kDaβ'-species, and the mature α- and β-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-dDa α'- and 83-kDa β'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the α'- and β'-species by sialic acid occurs, giving rise to the mature 135-kDa α- and 95-kDa β-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and preoteolytic cleavage to the α- and β (or α'- and β')-subunits. The 180-kDa species, which appears to be the aglyco-form of the 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin receptor.