Insulin-like growth factor-II (IGF-II) inhibits both the cellular uptake of β-galactosidase and the binding of β-galactosidase to purified IGF-II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor which targets acid hydrolases to lysosomes, has two different binding sites, one for the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal enzymes and the other for insulin-like growth factor-II (IGF-II). We have asked whether IGF-II can regulate the cellular uptake of the lysosomal enzyme 125I-β-galactosidase by modulating the binding of 125I-β-galactosidase to the IGF-II/Man-6-P receptor. We first isolated high affinity 125I-β-galactosidase by affinity chromatography on an IGF-II/Man-6-P receptor-Sepharose column. Specific uptake (mannose 6-phosphate-inhibitable) of 125I-β-galactosidase in BRL 3A2 rat liver cells and in rat C6 glial cells was 3.7–4.8 and 4.0–8.0% of added tracer, respectively. The cell-associated 125I-β-galactosidase in the uptake experiments largely represented internalized radioligand as measured by acid or mannose 6-phosphate washing. The uptake of 125I-β-galactosidase was inhibited by an antiserum (No. 3637) specific for the IGF-II/Man-6-P receptor. Low concentrations of IGF-II also inhibited the uptake of 125I-β-galactosidase. Maximal concentrations of IGF-II inhibited uptake by 73 ± 8% (mean ± S.D.) in C6 cells and by 77 ± 6% in BRL 3A2 cells compared to the level of inhibition by mannose 6-phosphate. The relative potency of IGF-II, IGF-I, and insulin (IGF-II ≫ IGF-I; insulin, inactive) were characteristic of the relative affinities of the ligands for the IGF-II/Man-6-P receptor. IGF-II also partially inhibited the binding of 125I-β-galactosidase to C6 and BRL 3A2 cells at 4 ° C and inhibited the binding to highly purified IGF-II/Man-6-P receptor by 58 ± 14%. We conclude that IGF-II inhibits the cellular uptake of 125I-β-galactosidase and that this inhibition is partly explained by the ability of IGF-II to inhibit binding of 125I-β-galactosidase to the IGF-II/Man-6-P receptor.