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PMID: 8415674 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 90 ·No. 19 ·1993-10-01 ·Pages 9176-80

Kozasa T, Hepler JR, Smrcka AV, Simon MI, Rhee SG, Sternweis PC, Gilman AG

Abstract

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.

MeSH Terms
Aluminum/pharmacology Animals Cell Line Chromatography, Ion Exchange Cloning, Molecular Enzyme Activation Fluorides/pharmacology GTP-Binding Proteins/isolation & purification,metabolism Gene Transfer Techniques Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Humans Isoenzymes/isolation & purification,metabolism Kinetics Macromolecular Substances Magnesium/pharmacology Moths Recombinant Proteins/isolation & purification,metabolism Restriction Mapping Tumor Cells, Cultured Type C Phospholipases/isolation & purification,metabolism
Chemicals
Isoenzymes Macromolecular Substances Recombinant Proteins Guanosine 5'-O-(3-Thiotriphosphate) Aluminum Type C Phospholipases GTP-Binding Proteins Magnesium Fluorides
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Kozasa T
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Hepler J R
Smrcka A V
Simon M I
Rhee S G
Sternweis P C
Gilman A G
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.90.19.9176
Published
1993-10-01
Pages
9176-80
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC47525
Subset
IM
Grants
NIGMS NIH HHS · GM31954 · United States
NIGMS NIH HHS · GM34236 · United States
NIGMS NIH HHS · GM34497 · United States
External Links
PubMed source DOI: 10.1073/pnas.90.19.9176 PMC Full Text View journal details
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