Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation.
Myristoylated G(o)alpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G(o)alpha) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G(o)alpha, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5;-3-O-(thio)triphosphate (GTPgammaS) activation induced disaggregation of the G(o)alpha oligomers to monomers. The G(o)alpha was crosslinked by a cross-linker, N,N-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G(o)alpha was pretreated with GTPgammaS. Immunoblot analysis also demonstrated oligomerization of the purified G(o)alpha proteins and its disaggregation triggered by GTPgammaS. These results provided direct evidence for the "disaggregation-coupling" theory and the disaggregation action of GTPgammaS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.
Zhang
L
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
Yang
S
S
Huang
Y
Y
D015153:Blotting, Western; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008301:Maleimides; D019814:Myristic Acid / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction
D003432:Cross-Linking Reagents; D008301:Maleimides; D019814:Myristic Acid; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C011328:N,N'-4-phenylenedimaleimide; D019206:GTP-Binding Protein alpha Subunits, Gi-Go