Transforming Growth Factor-β Induces Formation of a Dithiothreitol-resistant Type I/Type II Receptor Complex in Live Cells

doi:10.1074/jbc.274.9.5716

Transforming Growth Factor-β Induces Formation of a Dithiothreitol-resistant Type I/Type II Receptor Complex in Live Cells*

From ^tObThe Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, ^tOeDepartment of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, ^tOaDepartment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, and ⁱDepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Abstract

Transforming growth factor-β (TGF-β) binds to and signals via two serine-threonine kinase receptors, the type I (TβRI) and type II (TβRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TβRI and TβRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TβRI and TβRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TβRI·TβRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-β binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-β to TβRI, does not prevent formation of the TβRI·TβRII complex, but increases its sensitivity to detergent and prevents TGF-β-activated TβRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TβRI·TβRII complex, disrupted by dithiothreitol, or direct binding of TGF-β to TβRI is required for signaling.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA63260 (to H. F. L.) and DK02290 (to R. G. W.) and by grants from the Israel Science Foundation administered by the Israel Academy of Arts and Sciences and from the Israel Cancer Research Fund (to Y. I. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNc These authors contributed equally to this work.
↵FNd Current address: Dept. of Medicine, Yale School of Medicine, P. O. Box 208019, New Haven, CT 06520.
↵FNf Recipient of a fellowship from the Clore Scholars Programme.
↵FNg Supported by a postdoctoral fellowship from the Robert Steel Foundation for Pediatric Cancer Research.
↵FNh Supported by a National Institutes of Health postdoctoral fellowship.
↵FNj To whom correspondence should be addressed: The Whitehead Institute for Biomedical Research, 9 Cambridge Ctr., Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-6768; E-mail:lodish{at}wi.mit.edu.
↵2 R. Perlman and R. A. Weinberg, personal communication.
↵3 C. Rodriguez, R. Lin, R. G. Wells, P. Scherer, and H. F. Lodish, manuscript in preparation.
↵4 R. G. Wells and H. F. Lodish, unpublished results.
Abbreviations:

TGF-β

transforming growth factor-β

TβRI

TβRII, TβRIII, types I, II, and III TGF-β receptors

octyl-POE

n-octylpolyoxyethylene

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

MES

4-morpholineethanesulfonic acid

GST

glutathioneS-transferase

HA

hemagglutinin
- Received March 30, 1998.
- Revision received December 1, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.