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

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Abstract
Myristoylated Goα was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (Goα) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the Goα, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPγS) activation induced disaggregation of the Goα oligomers to monomers. The Goα 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 Goα was pretreated with GTPγS. Immunoblot analysis also demonstrated oligomerization of the purified Goα proteins and its disaggregation triggered by GTPγS. These results provided direct evidence for the “disaggregation–coupling” theory and the disaggregation action of GTPγS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.

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