Evidence for disaggregation of oligomeric G_oalpha induced by guanosine-5'-3-o-(thio)triphosphate activation

Myristoylated G_oalpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_oalpha) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_oalpha, 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_oalpha oligomers to monomers. The G_oalpha 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_oalpha was pretreated with GTPgammaS. Immunoblot analysis also demonstrated oligomerization of the purified G_oalpha 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.