TY - JOUR
T1 - Purification and characterization of recombinant G16α from Sf9 cells
T2 - Activation of purified phospholipase C isozymes by G-protein α subunits
AU - Kozasa, Tohru
AU - Hepler, John R.
AU - Smrcka, Alan V.
AU - Simon, Melvin I.
AU - Rhee, Sue Goo
AU - Sternweis, Paul C.
AU - Gilman, Alfred G.
PY - 1993/10/1
Y1 - 1993/10/1
N2 - A cDNA encoding G16α, the α subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16α in membrane extracts of Sf9 cells activated phospholipase C-β1 (PLC-β1) in the presence of guanosine 5′-[γ-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16α in the cytosolic fraction of Sf9 cells did not stimulate PLC-β1. Concurrent expression of the G-protein βγ subunit complex increased the amount of G16α in Sf9 cell membranes. The guanosine 5′-[γ-thio]triphosphate-activated form of G16α was purified from cholate extracts of membranes from cells expressing G16α, and the G-protein β2 and γ2 subunits. G16α activated PLC-β1, PLC-β2, and PLC-β3 in a manner essentially indistinguishable from that of Gqα. G16α-mediated activation of PLC-β1 and PLC-β3 greatly exceeded that of PLC-β2. G16α did not activate PLC-γ1 or PLC-δ. Thus, two distantly related members of the Gqα family, Gqα and G16α, have the same ability to activate the known isoforms of PLC-β.
AB - A cDNA encoding G16α, the α subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16α in membrane extracts of Sf9 cells activated phospholipase C-β1 (PLC-β1) in the presence of guanosine 5′-[γ-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16α in the cytosolic fraction of Sf9 cells did not stimulate PLC-β1. Concurrent expression of the G-protein βγ subunit complex increased the amount of G16α in Sf9 cell membranes. The guanosine 5′-[γ-thio]triphosphate-activated form of G16α was purified from cholate extracts of membranes from cells expressing G16α, and the G-protein β2 and γ2 subunits. G16α activated PLC-β1, PLC-β2, and PLC-β3 in a manner essentially indistinguishable from that of Gqα. G16α-mediated activation of PLC-β1 and PLC-β3 greatly exceeded that of PLC-β2. G16α did not activate PLC-γ1 or PLC-δ. Thus, two distantly related members of the Gqα family, Gqα and G16α, have the same ability to activate the known isoforms of PLC-β.
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U2 - 10.1073/pnas.90.19.9176
DO - 10.1073/pnas.90.19.9176
M3 - Article
C2 - 8415674
AN - SCOPUS:0027489221
SN - 0027-8424
VL - 90
SP - 9176
EP - 9180
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
ER -