The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-β receptor

Abstract

The type V transforming growth factor β (TGF-β) receptor (TβR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TβR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TβR·V in the presence of [γ-32P]ATP and MnCl2 with an apparent K(m) of 130 nM. TGF-β stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 °C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-β stimulated Man-6-P/IGF-II receptor-mediated uptake (~2-fold after 12 h treatment) of exogenous β- glucuronidase in Mv1Lu cells and type II TGF-β receptor (TβR-II)-defective mutant cells (DR26 cells) but not in type I TGF-β receptor (TβR-I)- defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TβR-I and TβR-II but lacking TβR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TβR-V and that both TβR-V and TβR-I may play a role in mediating the TGF-β-stimulated uptake of exogenous β-glucuronidase.