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 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.
Baek
K J
KJ
Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Das
T
T
Gray
C D
CD
Desai
S
S
Hwang
K C
KC
Gacchui
R
R
Ludwig
M
M
Im
M J
MJ
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*
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