Evidence for Disaggregation of Oligomeric G_oalpha Induced by Guanosine-5´-3-O-(thio)triphosphate Activation

L. Zhang, S. Yang, and Y. Huang^*

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; fax: 0086-010-64872026; E-mail: huang@sun5.ibp.ac.cn

^* To whom correspondence should be addressed.

Received March 16, 2002; Revision received April 25, 2002

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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.

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KEY WORDS: G_oalpha, GTPgammaS, chemical cross-linking, oligomerization and disaggregation, GDP/GTP exchange

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