A 50 KDA PROTEIN MODULATES GUANINE-NUCLEOTIDE-BINDING OF TRANSGLUTAMINASE-II

Regulation of cellular response is an important mechanism for controll ing cellular functions. The transmembrane signaling of the hormone rec eptors is regulated by GTP-binding proteins (GTPases) and their associ ated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, G alpha(h) (transglutaminase II), consistently c opurified with an similar to 50 kDa protein (G beta(h)) which is disso ciated 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 alp ha(h)-associated protein and down-regulates functions of G alpha h. Th us, polyclonal antibody against G beta(h) coimmunoprecipitates GDP-bou nd G alpha h but not the GDP-A1F(4)(-)bound form. The GTP gamma S bind ing and GTPase activity of G alpha(h) are inhibited in a G beta(h) con centration dependent manner. Supporting this notion, G beta(h) acceler ates 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 e xhibits TGase activity that is inhibited in the presence of the alpha( 1)-agonist and GTP. The GTP gamma S binding by the ternary complex, co nsisting 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 c omplex requires a greater than or equal to 2.7-fold higher concentrati on of G beta(h) than that for G alpha(h) alone, indicating that the re ceptor enhances the affinity of G alpha(h) for GTP. In addition, G bet a(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 a lpha(1)-adrenoceptor. These findings clearly demonstrate that G alpha( h) associates with a novel protein which modulates the affinity of G a lpha(h) for guanine nucleotides and that the GDP-bound G(h) is the gro und state for the counterpart activator, the alpha(1)-adrenoceptor, in this signaling system.