A 50 kDa protein modulates guanine nucleotide binding of transglutaminase II

Regulation of cellular response is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins (GTPases) and their associated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, G alpha(h) (transglutaminase II), consistently copurified with an similar to 50 kDa protein (G beta(h)) which is dissociated from G alpha(h) upon activation with GTP gamma S or AIF(4)(-). Present immunological and biochemical studies on the regulation of the GTPase cycle of G alpha(h), which involves the alpha(1)-adrenoceptor and 50 kDa G beta(h), reveal that the 50 kDa protein is indeed a G alpha(h)-associated protein and down-regulates functions of G alpha h. Thus, polyclonal antibody against G beta(h) coimmunoprecipitates GDP-bound G alpha h but not the GDP-A1F(4)(-)bound form. The GTP gamma S binding and GTPase activity of G alpha(h) are inhibited in a G beta(h) concentration dependent manner. Supporting this notion, G beta(h) accelerates GTP gamma S release from G alpha(h) and changes the affinity of G alpha(h) from GTP to GDP. Moreover, the ternary complex preparation exhibits TGase activity that is inhibited in the presence of the alpha(1)-agonist and GTP. The GTP gamma S binding by the ternary complex, consisting of the al-agonist, the receptor, and G(h), is also inhibited by G beta(h). The inhibition of GTP gamma S binding with the ternary complex requires a greater than or equal to 2.7-fold higher concentration of G beta(h) than that for G alpha(h) alone, indicating that the receptor enhances the affinity of G alpha(h) for GTP. In addition, G beta(h) copurifies with an alpha(1)-agonist, adrenoceptor, and G alpha(h) ternary . complex, showing that the complex is a heterotetramer. Our data also suggest that G beta(h) does not directly interact with the alpha(1)-adrenoceptor. These findings clearly demonstrate that G alpha(h) associates with a novel protein which modulates the affinity of G alpha(h) for guanine nucleotides and that the GDP-bound G(h) is the ground state for the counterpart activator, the alpha(1)-adrenoceptor, in this signaling system.