CHARACTERIZATION OF TRANSFORMING GROWTH-FACTOR-BETA (TGF-BETA) RECEPTORS ON BEWO CHORIOCARCINOMA CELLS INCLUDING THE IDENTIFICATION OF A NOVEL 38-KDA TGF-BETA BINDING GLYCOPROTEIN

Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta1 and TGF-beta2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta1 and TGF-beta2. The K(d) values obtained from Scatchard analyses were approximately 65 pM for I-125-TGF-beta1 and approximately 40 pM for I-125-TGF-beta2, with 70 000 and 85 000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta1 and TGF-beta2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with I-125-TGF-beta1 and I-125-TGF-beta2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta1 than for -beta2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta2 than for -beta1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta2 than for -beta1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.