Identification of important regions in the cytoplasmic juxtamembrane domain of type I receptor that separate signaling pathways of transforming growth factor-β

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 era 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.