title: A 50 KDa protein modulates guanine nucleotide binding of transglutaminase II.
abstract: 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 approximately 50 kDa protein (G Beta h) which is dissociated from G alpha h upon activation with GTP gamma S or AlF4-. 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-AlF4--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 accelerated 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 alpha 1-agonist, the receptor, and Gh, is also inhibited by G beta h. The inhibition of GTP gamma S binding with the ternary complex requires a > or = 2.7-fold higher concentration of G beta h than the 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 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 Gh is the ground state for the counterpart activator, the alpha 1-adrenoceptor, in this signaling system.
authors.affiliation: Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Baek
authors.forename: T
authors.initials: T
authors.lastname: Das
authors.forename: C D
authors.initials: CD
authors.lastname: Gray
authors.forename: S
authors.initials: S
authors.lastname: Desai
authors.forename: K C
authors.initials: KC
authors.lastname: Hwang
authors.forename: R
authors.initials: R
authors.lastname: Gacchui
authors.forename: M
authors.initials: M
authors.lastname: Ludwig
authors.forename: M J
authors.initials: MJ
authors.lastname: Im
mesh_terms: D000818:Animals; D015536:Down-Regulation; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006150:Guanine Nucleotides; D011506:Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011503:Transglutaminases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins