A pivotal role for the transmembrane domain in transforming growth factor-β receptor activation

Transforming growth factor-beta (TGF-beta) delivers diverse growth and differentiation signals by binding two distantly related transmembrane serine/threonine kinase receptors: the type I receptor (T beta RI) and the type II receptor (T beta RII), In an attempt to establish the role of the transmembrane domain in receptor signaling, two chimeric TGF-beta receptors, T beta RI-II-I and T beta RII-I-II, containing the opposite transmembrane domain were generated. When transfected into a mutant mink lung epithelial cell line R1B, which lacks functional T beta RI, T beta RI-II-I restored TGF-beta 1-induced transcriptional activation of a TGF-beta reporter p3TP-Lux to similar to 25% of the levels restored by wild-type T beta RI, In the mutant mink lung epithelial cell line DR26, which contains a truncated, nonfunctional T beta RII, wild-type receptor T beta RII restored the TGF-beta responsiveness, while the T beta RII-I-II cDNA was inactive, When both T beta RI and T beta RII were transfected into R1B, DR26, or Mv1Lu cells, a low level of constitutive p3TP-Lux activity was observed. However, cotransfection of both transmembrane chimeric receptors, T beta RI-II-I and T beta RII-I-II, or the wild-type T beta RI with the transmembrane chimeric T beta RII-I-II resulted in high levels of ligand-independent receptor activation. These results suggest that the transmembrane domains of both TGF-beta receptors are essential and play a pivotal role in receptor activation. To investigate the role of the transmembrane domain further, four type II transmembrane mutants were generated: T beta RII Delta-1, T beta RII Delta-2, T beta RII Delta-3, and T beta RII Delta-4, which have one, two, three, or four amino acids deleted at the N terminus of the transmembrane domain, respectively. Interestingly, co expression of T beta RII Delta-1 with the wild-type T beta RI in DR26 cells resulted in high levels of constitutive activation, while only low levels of the activation were observed when T beta RII Delta-2, T beta RII Delta-3, or T beta RII Delta-4 were co-expressed with the wildtype T beta RI, However, T beta RII Delta-1 restored very little the TGF-beta responsiveness in DR26cells, Expression of T beta RII Delta-2, T beta RII Delta-3, and T beta RII Delta-4 resulted in a progressive increase in TGF-P responsiveness, with T beta RII Delta-4 reaching the level of activity of the wild-type T beta RII, Furthermore, like T beta RII-I-II, co-expression of T beta RII Delta-1 with T beta RI-II-I also resulted in high levels of constitutive activation. These results are consistent with an important role for the transmembrane region of the receptors, We further propose a model of receptor activation in which receptor activation occurs via relative orientational rotation.