A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor β receptor

Transforming growth factor beta (TGF beta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGF beta receptor (T beta R-II) phosphorylating type I TGF beta receptor (T beta R-I). Because the failure of interaction cloning, thus far, to identify bona fide T beta R-I substrates might reasonably have been due to the use of inactive T beta R-I as bait, we sought to identify molecules that interact specifically with active T beta R-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 TGF beta signaling we have previously disproved; the charge substitution at Thr(204) constitutively activates T beta R-I. Unlike previous screens using mild-type T beta R-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, T beta R-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated T beta R-I, but not wild-type T beta R-I, T beta R-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated T beta R-I and ligand-activated T beta R-I, but not wild-type T beta R-I in the absence of TGF beta. The partial TRAP-I protein that specifically binds these mutationally and ligand-activated farms of T beta R-I can inhibit signaling by the native receptor after stimulation with TGF beta or by the constitutively activated receptor mutation, as measured by a TGF beta-dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGF beta and may potentially have a functional role in TGF beta signaling.