Myristoylated Goalpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (Goalpha) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the Goalpha, 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 Goalpha oligomers to monomers. The Goalpha 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 Goalpha was pretreated with GTPgammaS. Immunoblot analysis also demonstrated oligomerization of the purified Goalpha 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.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Jan 1 2003|
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