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.

Abstract

Transforming growth factor-: (TGF-f) 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-41 and TGF-f2 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 TGFf1 and TGF-,32. The Kd values obtained from Scatchard analyses were -65 pM for 125I1 TGF-31 and -40 pM for 1251-TGF-fl2, with 70 000 and 85 000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-f1 and TGF-32 were equipotent (apparent half maximal inhibition [IC50] -70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 1251-TGF-l1 and 1251-TGF-32 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-f binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfatepolyacrylamide 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-31 than for -f2 yet exhibits a 5-to 10-fold higher affinity for TGF-,B2 than for -41. The 38-kDa TGF-1 binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-,B2 than for -f1 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.

Mapping of the ligand binding domain of the transforming growth factor beta receptor type III by deletion mutagenesis.

“…The first is a diffuse 400-to 150-kDa band that is characteristic of receptor type III. The other is a 50-kDa band that, due to its binding characteristics, was suggested by us to be a proteolytic fragment of receptor type III (34). We have now observed that both bands are specifically immunoprecipitated by an anti-peptide antibody directed against the C-terminal cytoplasmic domain of receptor type III (not shown).…”

Estrogen Receptor Expression and Growth-Promoting Function in Human Choriocarcinoma Cells

“…The mechanisms by which estrogens mediate their mitogenic effects on choriocarcinoma cell growth are unknown. However, the placenta expresses a variety of growth factors, including insulin growth factor-1 (IGF-1), transforming growth factor-ß (TGF-/3), and epidermal growth factor (EGF) and their respective binding proteins (Bhaumick et al, 1987;Morrish et al, 1987;Chen et al, 1990;Ringler and Strauss, 1990;Mitchell et al, 1992;Cao et al, 1994) and these growth factors play important roles in placental trophoblast function. For example, EGF promotes differentiation and secretion of hCG and hCS in trophoblastic cells (Marao et al, 1986;Morrish et al, 1987) and IGF-1 increases hCS production in term placenta (Bhaumick et al, 1987).…”

TGF‐β control of cell proliferation

“…Multiple cell surface receptors of various sizes have been identified in cultured cells and tissues by cross-linking 125 I-labeled-TGF-b ( 125 I-TGF-b) to these molecules in the presence of bifunctional cross-linking reagents. These include type I (TbR-I, MW$53,000), type II (TbR-II, MW$70,000), type III (TbR-III, MW$ 280,000-370,000), type IV (TbR-IV, MW$ 60,000), type V (TbR-V, MW$400,000), and type VI (TbR-VI, MW$180,000) receptors as well as several membrane-associated binding proteins (MW$38,000) [O'Grady et al, 1991a;Mitchell et al, 1992;Segarini, 1993;Massague, 1998;Moustakas et al, 2002]. TbR-I and TbR-II are Ser/Thr-specific protein kinases and are believed to be primarily responsible for TGF-b-induced cellular responses [Heldin et al, 1997;Massague, 1998;Roberts, 1998].…”

“…TbR-V coexpresses with TbR-I, TbR-II, and TbR-III in all normal cell types studied thus far [O'Grady et al, 1991a,b] and also serves as the insulin-like growth factor binding protein-3 (IGFBP-3) receptor, mediating IGF-independent growth inhibition by IGFBP-3 in responsive cells [Leal et al, 1997[Leal et al, , 1999Wu et al, 2000]. TbR-VI and other membrane-associated TGF-b binding proteins are expressed only in specific cell types [Mitchell et al, 1992;Segarini, 1993].…”