TY - JOUR
T1 - Evidence for disaggregation of oligomeric G0α induced by guanosine-5′-3-O-(thio)triphosphate activation
AU - Zhang, L.
AU - Yang, S.
AU - Huang, Y.
N1 - Funding Information:
This work was generously supported by Project 39730130 from the National Natural Science Foundation of China. We thank Professor Susanne Mumby (University of Texas, Southwestern Medical Center) for a gift of plasmid pQE60 (Goα) and Professor Gordon (University of Washington) for a gift of plasmid pBB131 (NMT).
PY - 2003/1
Y1 - 2003/1
N2 - Myristoylated G0α was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G0α) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G0α, 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 G0α oligomers to monomers. The G0α 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 G0α was pretreated with GTPγS. Immunoblot analysis also demonstrated oligomerization of the purified G0α 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.
AB - Myristoylated G0α was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G0α) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G0α, 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 G0α oligomers to monomers. The G0α 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 G0α was pretreated with GTPγS. Immunoblot analysis also demonstrated oligomerization of the purified G0α 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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U2 - 10.1023/A:1022161922325
DO - 10.1023/A:1022161922325
M3 - Article
C2 - 12693986
AN - SCOPUS:0037239967
SN - 0006-2979
VL - 68
SP - 121
EP - 128
JO - Biochemistry (Moscow)
JF - Biochemistry (Moscow)
IS - 1
ER -