Insulin-like growth factor-II (IGF-II) inhibits both the cellular uptake of beta-galactosidase and the binding of beta-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-beta-galactosidase by modulating the binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor. We first isolated high affinity 125I-beta-galactosidase by affinity chromatography on an IGF-II/Man-6-P receptor-Sepharose column. Specific uptake (mannose 6-phosphate-inhibitable) of 125I-beta-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-beta-galactosidase in the uptake experiments largely represented internalized radioligand as measured by acid or mannose 6-phosphate washing. The uptake of 125I-beta-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-beta-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 much greater than 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-beta-galactosidase to C6 and BRL 3A2 cells at 4 degrees 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-beta-galactosidase and that this inhibition is partly explained by the ability of IGF-II to inhibit binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor.
Kiess
W
W
Metabolism Branch, National Cancer Institute, Bethesda, Maryland 20892.
Thomas
C L
CL
Greenstein
L A
LA
Lee
L
L
Sklar
M M
MM
Rechler
M M
MM
Sahagian
G G
GG
Nissley
S P
SP
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002846:Chromatography, Affinity; D005260:Female; D005696:Galactosidases / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*
D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II; D005696:Galactosidases; D001616:beta-Galactosidase