Binding and internalization of transforming growth factor-beta 1 by human hepatoma cells: evidence for receptor recycling.
Cellular processing of 125I-labeled transforming growth factor-beta 1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-beta 1 to cell surface receptors was specific, saturable and calcium-independent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 x 10(-10) mol/L) binding sites (4.5 x 10(3) for the Hep G2 cell; 1.5 x 10(3) for the Hep 3B cell) for both human and porcine transforming growth factor-beta 1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-beta 1 was removed by brief exposure to acidic medium (pH less than 4) but was converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Spontaneous dissociation of bound ligand over a 6 hr period at 4 degrees C was less than 10%. Disuccinimidyl suberate was used to covalently label 125I-transforming growth factor-beta 1 to cell-surface binding sites. Labeling of the ligand/receptor complexes was inhibited by unlabeled transforming growth factor-beta 1 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-beta 1 was internalized and degraded at 37 degrees C, and the products were released into the medium as trichloroacetic acid-nonprecipitable radioactivity. The lysosomotropic base chloroquine and the carboxylic ionphore monensin inhibited degradation and release of 125I-labeled products from the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Sathre
K A
KA
Department of Medicine, University of Minnesota, Minneapolis 55455.
Tsang
M L
ML
Weatherbee
J A
JA
Steer
C J
CJ
D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002738:Chloroquine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008985:Monensin / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000502:physiology*; D018125:Receptors, Transforming Growth Factor beta; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D011956:Receptors, Cell Surface; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D002738:Chloroquine; D008985:Monensin; D003513:Cycloheximide