Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.
Kozasa
T
T
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Hepler
J R
JR
Smrcka
A V
AV
Simon
M I
MI
Rhee
S G
SG
Sternweis
P C
PC
Gilman
A G
AG
D000535:Aluminum / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D018014:Gene Transfer Techniques; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009036:Moths; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000535:Aluminum; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D008274:Magnesium; D005459:Fluorides