Biosynthesis of the insulin-like growth factor-II (IGF-II)/mannose-6-phosphate receptor in rat C6 glial cells: the role of N-linked glycosylation in binding of IGF-II to the receptor.

We examined the role of N-linked glycosylation of the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (Man-6-P) receptor in binding of 125I]IGF-II to the receptor. First we studied the synthesis and posttranslational processing of this receptor in rat C6 glial cells, which have abundant IGF-II/Man-6-P receptors. Cells were pulse labeled with 35S]methionine and lysed, and the IGF-II/Man-6-P receptor was immunoprecipitated using a specific IGF-II/Man-6-P receptor antibody (no. 3637). Analysis of the immunoprecipitate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reduction of disulfide bonds showed a 235-kDa receptor precursor that was processed into the mature 245-kDa IGF-II/Man-6-P receptor within 2 h of chase. Digestion of the 235-kDa precursor with endoglycosidase-H (Endo H) produced a 220-kDa form, whereas the mature 245-kDa receptor was relatively resistant to cleavage by Endo H. When cells were cultured in the presence of 2 microM monensin, the 235-kDa receptor was not further processed into the mature Endo H-resistant receptor form. In addition, the presence of swainsonine in C6 glial cell cultures led to the formation of a 240-kDa receptor hybrid molecule, which was cleaved by Endo H into a 225-kDa species. When tunicamycin was present during the pulse-chase labeling experiment, a 220-kDa receptor species accumulated. This species was 205 kDa by immunoblotting when SDS-PAGE was performed under nonreducing conditions. Pure IGF-II/Man-6-P receptor was digested with N-glycosidase-F, and the digest was immunoblotted with antiserum 3637 after SDS-PAGE under nonreducing conditions. Whereas undigested receptor was a single band of 215 kDa under nonreducing conditions, digested receptor was 205 kDa. The binding affinity of IGF-II for the digested receptor was the same as the binding affinity of IGF-II for the undigested receptor. In addition, affinity cross-linking experiments showed that 125I]IGF-II also bound to the unglycosylated receptor precursor that accumulated in the tunicamycin-treated cells, and the binding affinity of IGF-II for this species was indistinguishable from the binding affinity of IGF-II for the mature receptor. We conclude that IGF-II can bind to an IGF-II/Man-6-P receptor that lacks N-linked oligosaccharides.