G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation.
The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.
Dingus
Jane
J
Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Wells
Christopher A
CA
Campbell
Lia
L
Cleator
John H
JH
Robinson
Kathryn
K
Hildebrandt
John D
JD
D000818:Animals; D002478:Cells, Cultured; D019281:Dimerization; D000939:Epitopes / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D054799:Phospholipase C beta; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D000939:Epitopes; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins