Petra Franzen

Cloning of a TGFβ type I receptor that forms a heteromeric complex with the TGFβ type II receptor

A cDNA clone encoding a 53 kd serine/threonine kinase receptor with an overall structure similar to that of the type II receptor for transforming growth factor beta (TGF beta) was obtained. 125I-TGF beta 1 bound to porcine endothelial cells transfected with the cDNA and formed a cross-linked complex of 70 kd, characteristic of a TGF beta type I receptor. Immunoprecipitation of the cross-linked complexes by antibodies against the cloned receptor revealed the 70 kd complex as well as a 94 kd TGF beta type II receptor complex. The immunoprecipitated novel serine/threonine kinase receptor had biochemical properties of the TGF beta type I receptor and was observed in different cell types. Transfection of the cloned cDNA into TGF beta type I receptor-deficient cells restored TGF beta-induced plasminogen activator inhibitor 1 production. These results suggest that signal transduction by TGF beta involves the formation of a heteromeric complex of two different serine/threonine kinase receptors.

A novel GTPase-activating protein for Rho interacts with a PDZ domain of the protein-tyrosine phosphatase PTPL1.

PTPL1 is an intracellular protein-tyrosine phosphatase that contains five PDZ domains. Here, we present the cloning of a novel 150-kDa protein, the four most C-terminal amino acid residues of which specifically interact with the fourth PDZ domain of PTPL1. The molecule contains a GTPase-activating protein (GAP) domain, a cysteine-rich, putative Zn2+- and diacylglycerol-binding domain, and a region of sequence homology to the product of the Caenorhabditis elegans geneZK669.1a. The GAP domain is active on Rho, Rac, and Cdc42in vitro but with a clear preference for Rho; we refer to the molecule as PTPL1-associated RhoGAP 1, PARG1. Rho is inactivated by GAPs, and protein-tyrosine phosphorylation has been implicated in Rho signaling. Therefore, a complex between PTPL1 and PARG1 may function as a powerful negative regulator of Rho signaling, acting both on Rho itself and on tyrosine phosphorylated components in the Rho signal transduction pathway.

Serine/threonine kinase receptors

A new family of transmembrane receptors that contain intracellular serine/threonine kinase domains is emerging. Ligands for this class of receptors include members of the transforming growth factor-beta (TGF-beta) superfamily, e.g. TGF-beta s and activins. TGF-beta s exert their effects on target cells via formation of heteromeric serine/threonine kinase complexes (TGF-beta type I and type II receptors). Other components, i.e. TGF-beta type III receptor and endoglin, appear to have more indirect roles, e.g. to present ligands to the signalling receptors. Given the structural similarity between members of the TGF-beta superfamily, other ligands in this family may act through structurally and functionally similar serine/threonine kinase receptors.

Transforming Growth Factor (TGF-β)-specific Signaling by Chimeric TGF-β Type II Receptor with Intracellular Domain of Activin Type IIB Receptor

Members of the transforming growth factor-β (TGF-β) superfamily signal via different heteromeric complexes of two sequentially acting serine/threonine kinase receptors, i.e.type I and type II receptors. We generated two different chimeric TGF-β superfamily receptors, i.e. TβR-I/BMPR-IB, containing the extracellular domain of TGF-β type I receptor (TβR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TβR-II/ActR-IIB, containing the extracellular domain of TGF-β type II receptor (TβR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-β1, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed heteromeric complexes with wild-type TβR-II and TβR-I, respectively, upon stable transfection in mink lung epithelial cell lines. We show that TβR-II/ActR-IIB restored the responsiveness upon transfection in mutant cell lines lacking functional TβR-II with respect to TGF-β-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation. Moreover, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed a functional complex in response to TGF-β and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TβR-I/BMPR-IB to restore responsiveness to TGF-β in cell lines deficient in functional TβR-I. The fact that the TGF-β1-induced complex between TβR-II/ActR-IIB and TβR-I stimulated endogenous Smad2 phosphorylation, a TGF-β-like response, is in agreement with the current model for receptor activation in which the type I receptor determines signal specificity.

Characterization of a 60-kDa cell surface-associated transforming growth factor-beta binding protein that can interfere with transforming growth factor-beta receptor binding

We have characterized a 60‐kDa transforming growth factor‐β (TGF‐β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross‐linking of cell surface proteins by using 125I‐TGF‐β1. Binding of 125I‐TGF‐β1 to the 60‐kDa protein was competed by an excess of unlabeled TGF‐β1 but not by TGF‐β2, TGF‐β3, activin, or osteogenic protein‐1 (OP‐1), also termed bone morphogenetic protein‐7 (BMP‐7). In addition, no binding of 125I‐TGF‐β2 and 125I‐TGF‐β3 to the 60‐kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60‐kDa TGF‐β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF‐β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60‐kDa protein from the cell surface. In addition, we found that the previously described TGF‐β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF‐β receptors, could be released from the cell surface by these same treatments. This suggests that the 60‐kDa protein and the similarly sized TGF‐β type IV receptor are related proteins. The eluted 60‐kDa LNCaP protein was shown to interfere with the binding of TGF‐β to the TGF‐β receptors. Thus, the cell surface‐associated 60‐kDa TGF‐β binding protein may play a role in regulating TGF‐β binding to TGF‐β receptors. J. Cell. Physiol. 173:447–459, 1997.