Binding and internalization of transforming growth factor-β1 by human hepatoma cells: Evidence for receptor recycling

Abstract

Cellular processing of 125I-labeled transforming growth factor-β1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-β1 to cell surface receptors was specific, saturable and calciumindependent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 × 10−10 mol/L) binding sites (4.5 × 103 for the Hep G2 cell; 1.5 × 103 for the Hep 3B cell) for both human and porcine transforming growth factor-β1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-β1 was removed by brief exposure to acidic medium (pH <4) but was converted into an acid-resistant state rapidly after shifting the cells to 37°C. Spontaneous dissociation of bound ligand over a 6 hr period at 4° C was less than 10%. Disuccinimidyl suberate was used to covalently label 125I-transforming growth factor-β1 to cell-surface binding sites. Labeling of the ligand/receptor complexes was inhibited by unlabeled transforming growth factor-β1 but was unaffected by other growth factors. The radiolabeled complexes showed approximate molecular weights of 280,000, 85,000 and 65,000 when run on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell-bound 125I-transforming growth factor-β1 was internalized and degraded at 37° C, and the products were released into the medium as trichloroacetic acid-nonprecipitable radioactivity. The lysosomotropic base chloroquine and the carboxylic ionophore monensin inhibited degradation and release of 125I-labeled products from the cells. In the presence of cycloheximide and under conditions of sustained binding and uptake of saturating amounts of 125I-transforming growth factor-β1 for 3 hr, a 20% decrease in the binding capacity of Hep G2 cells occurred. The result indicates that during active processing of the 125I-transforming growth factor-β1 receptor complex by Hep G2 cells, surface receptors for transforming growth factor-β1 are replenished either from a cryptic intracellular pool or by receptor recycling. (HEPATOLOGY 1991;14:287–295.)