Interaction of insulin-like growth factor II with rat chondrocytes: receptor binding, internalization, and degradation.
We have characterized the interaction of insulin-like growth factor II (IGF-II) with its plasma membrane receptor(s) on cultured rat chondrocytes. Our studies, paralleling those already reported for IGF-I, demonstrate that [125I]IGF-II binds to these receptors with a high degree of affinity and that this process is reversible, specific, and time, temperature, and concentration dependent. At 4 C, unlabeled IGF-II causes half-maximal displacement of the labeled ligand at a concentration of 22 ng/ml, whereas IGF-I is approximately 1/200th as potent, and insulin does not displace [125I]IGF-II even at a concentration of 10 micrograms/ml. Maximum binding to chondrocytes (44% of added radioactivity) occurred after 4-5 h of incubation at 15 C. Compared to [125I]IGF-I binding, this value is 7-fold higher and is consistent with an affinity constant (Ka = 3.8 X 10(8) M-1) approximately 1 order of magnitude greater. Photoaffinity labeling studies disclose that IGF-II binds primarily to the type II IGF receptor, which has an apparent mol wt of 220K when electrophoresed under nonreducing conditions and 270K under reducing conditions. Nanomolar concentrations of IGF-II stimulated the synthesis of DNA and RNA in a dose-related manner, and micromolar concentrations of insulin demonstrated an additive effect with respect to the incorporation of [3H]thymidine into DNA, but not [3H]uridine into RNA. Preincubation of rat chondrocytes with increasing concentrations of insulin caused a marked dose-related increase in [125I]IGF-II binding, a phenomenon previously reported in several other cell types. In addition to defining the binding characteristics of IGF-II, we used the lysosomotropic agents chloroquine and ammonium chloride to demonstrate that its ligand-receptor complex, like that of IGF-I, is internalized and degraded partially via the lysosomal pathway.
D000345:Affinity Labels; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002356:Cartilage / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D007328:Insulin / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D010777:Photochemistry; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000378:metabolism*; D013696:Temperature
D000345:Affinity Labels; D007328:Insulin; D017451:Receptors, Somatomedin; D013002:Somatomedins; D000643:Ammonium Chloride; D012313:RNA; D007335:Insulin-Like Growth Factor II; D002738:Chloroquine; D004247:DNA; D011972:Receptor, Insulin