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

Kim A. Sathre, Monica L.‐S Tsang, James A. Weatherbee, Clifford J. Steer

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


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.)

Original languageEnglish (US)
Pages (from-to)287-295
Number of pages9
Issue number2
StatePublished - Aug 1991


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