Purification and characterization of recombinant G16α from Sf9 cells: Activation of purified phospholipase C isozymes by G-protein α subunits

Tohru Kozasa, John R. Hepler, Alan V. Smrcka, Melvin I. Simon, Sue Goo Rhee, Paul C. Sternweis, Alfred G. Gilman

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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 G. 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 G family, G and G16α, have the same ability to activate the known isoforms of PLC-β.

Original languageEnglish (US)
Pages (from-to)9176-9180
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number19
StatePublished - Oct 1 1993

ASJC Scopus subject areas

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