Identification of Important Regions in the Cytoplasmic Juxtamembrane Domain of Type I Receptor That Separate Signaling Pathways of Transforming Growth Factor-

doi:10.1074/jbc.271.5.2769

Identification of Important Regions in the Cytoplasmic Juxtamembrane Domain of Type I Receptor That Separate Signaling Pathways of Transforming Growth Factor-(*)

From the (1)Department of Oral Pathology and the
(2)First and
(3)Second Departments of Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan, the
(4)Department of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan, and the
(5)Ludwig Institute for Cancer Research, Biomedical Center, S-751 24 Uppsala, Sweden

^§To whom correspondence should be addressed:
Dept. of Biochemistry, The Cancer Institute, Tokyo, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan
. Tel.: 81-3-3918-0111, ext. 4504; Fax: 81-3-3918-0342.

Abstract

Proteins in the transforming growth factor-β (TGF-β) superfamily exert their effects by forming heteromeric complexes of their type I and type II serine/threonine kinase receptors. The type I and type II receptors form distinct subgroups in the serine/threonine kinase receptor family based on the sequences of the kinase domains and the presence of a highly conserved region called the GS domain (or type I box) located just N-terminal to the kinase domain in the type I receptors. Recent studies have revealed that upon TGF-β binding several serine and threonine residues in the GS domain of TGF-β type I receptor (TβR-I) are phosphorylated by TGF-β type II receptor (TβR-II) and that the phosphorylation of GS domain is essential for TGF-β signaling. Here we investigated the role of cytoplasmic juxtamembrane region located between the transmembrane domain and the GS domain of TβR-I by mutational analyses using mutant mink lung epithelial cells, which lack endogenous TβR-I. Upon transfection, wild-type TβR-I restored the TGF-β signals for growth inhibition and production of plasminogen activator inhibitor-1 (PAI-1) and fibronectin. A deletion mutant, TβR-I/JD1(Δ150-181), which lacks the juxtamembrane region preceding the GS domain, bound TGF-β in concert with TβR-II and transduced a signal leading to production of PAI-1 but not growth inhibition. Recombinant receptors with mutations that change serine 172 to alanine (S172A) or threonine 176 to valine (T176V) were similar to wild-type TβR-I in their abilities to bind TGF-β, formed complexes with TβR-II, and transduced a signal for PAI-1 and fibronectin. Similar to TβR-I/JD1(Δ150-181), however, these missense mutant receptors were impaired to mediate a growth inhibitory signal. These observations indicate that serine 172 and threonine 176 of TβR-I are dispensable for extracellular matrix protein production but essential to the growth inhibition by TGF-β.

Footnotes

↵* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and a grant from the Mochida Memorial Foundation For Medical and Pharmaceutical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGF-β

transforming growth factor-β

BMP

bone morphogenetic protein

TβR

TGF-β receptor

ActR

activin receptor

BMPR

BMP receptor

PAI

plasminogen activator inhibitor

PCR

polymerase chain reaction

GST

glutathione S-transferase

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

PBS

phosphate-buffered saline

DTT

dithiothreitol.
↵²H. Nishitoh, M. Saitoh, I. Asahina, S. Enomoto, T. K. Sampath, M. Takagi, and H. Ichijo, submitted for publication.
- Received August 21, 1995.
- Revision received November 20, 1995.