A Novel Protein Distinguishes between Quiescent and Activated Forms of the Type I Transforming Growth Factor β Receptor

Abstract Transforming growth factor β (TGFβ) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFβ receptor (TβR-II) phosphorylating type I TGFβ receptor (TβR-I). Because the failure of interaction cloning, thus far, to identify bona fide TβR-I substrates might reasonably have been due to the use of inactive TβR-I as bait, we sought to identify molecules that interact specifically with active TβR-I, employing the triple mutation L193A,P194A,T204D in a yeast two-hybrid system. The Leu-Pro substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGFβ signaling we have previously disproved; the charge substitution at Thr204 constitutively activates TβR-I. Unlike previous screens using wild-type TβR-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, TβR-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated TβR-I, but not wild-type TβR-I, TβR-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated TβR-I and ligand-activated TβR-I, but not wild-type TβR-I in the absence of TGFβ. The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of TβR-I can inhibit signaling by the native receptor after stimulation with TGFβ or by the constitutively activated receptor mutation, as measured by a TGFβ-dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFβ and may potentially have a functional role in TGFβ signaling.