Transforming growth factor (TGF)-β1 internalization -: Modulation by ligand interaction with TGF-β receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-p (TGF-P) internalization was studied by monitoring the uptake of I-125-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-P receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degreesC internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII, Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with I-125-TGF-beta1 at 37 degreesC indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degreesC for 2 h prior to switching the cells to 37 degreesC. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degreesC and by a reduction in the labeling intensities of RI and RII in I-125-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degreesC and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degreesC, suggesting that prebinding promotes formation of stable RI RII complexes that can signal independently of ligand.