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.
Kiess
W
W
Children's Hospital, University of Munich, Germany.
Greenstein
L A
LA
Lee
L
L
Thomas
C
C
Nissley
S P
SP
D000118:Acetylglucosaminidase / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D009457:Neuroglia / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000378:metabolism*; D017451:Receptors, Somatomedin; D014415:Tunicamycin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D011498:Protein Precursors; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D014415:Tunicamycin; D007335:Insulin-Like Growth Factor II; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase