G Protein βγ Dimer Formation:  Gβ and Gγ Differentially Determine Efficiency of in Vitro Dimer Formation

Abstract

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.

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

“…With the exception of the β 5 -subunit, all combinations of the other four β-subunits (β 1 -β 4 ) and 12 γ-subunits (γ 1 -γ 5 and γ 7 -γ 13 ) were found to interact with the two different α-subunits. This finding was unexpected because other investigators had indicated that certain βγ-dimers (e.g., β 2 γ 1 , β 2 γ 11 , β 2 γ 13 , β 3 γ 1 , and β 3 γ 11 ) do not exist (6,(26)(27)(28)(29)(30)(31)(32)(33)(34).…”

“…Previous βγ-dimerization studies have used various expression systems [e.g., in vitro translation (32,33), Sf9 cells (26), yeast (29), and mammalian cells (27,28,30,31,34)] and various dimerization assays [e.g., coimmunoprecipitation (30)(31)(32), yeast two-hybrid screens (29), membrane targeting of the β-subunit (27), purification of functional dimers (26,28), and bimolecular fluorescence complementation (a complementation of YFP from two fragments) (34)]. An important difference from other studies is our mode of expression.…”

“…Many βγ-dimers can be generated by simply mixing subunits that have been separately translated in vitro (reticulocyte lysate) (32), and this may be an indication that for the formation of those combinations there is no significant activation barrier, and thus they do not necessarily need extensive assistance during assembly. For example, DRiP78 is associated with the membrane of the endoplasmic reticulum and therefore is unlikely to be present in the reticulocyte lysate unless microsomal fractions are added additionally.…”

“…However, because combinations like β 2 γ 2 , β 3 γ 2 , β 4 γ 1 , or β 4 γ 2 are known to exist, it is hard to rationalize, based on the sequence differences analyzed, why combinations like β 3 γ 1 and especially β 2 γ 1 should be excluded per se, as had been proposed (6,(26)(27)(28)(29)(30)(31)(32)(33)(34). More likely, the differences between β 1 and β 2 or β 3 , especially if no α-subunit is bound, may result in slightly altered βγ-dimer interfaces and therefore slightly reduced affinities between these subunits.…”

“…More likely, the differences between β 1 and β 2 or β 3 , especially if no α-subunit is bound, may result in slightly altered βγ-dimer interfaces and therefore slightly reduced affinities between these subunits. The fact that β 3 , along with β 5 , is the β-subunit that least readily forms βγ-dimers (6,29,31,32), may be explained by the differences in interaction interface detailed above.…”