In vitro synthesis of G protein beta gamma dimers.
The guanine nucleotide-binding proteins (G proteins), which play a central role in coupling membrane-bound receptors to intracellular effectors, are heterotrimers composed of alpha, beta, and gamma subunits. The beta and gamma subunits form a functional monomer that does not appear to separate under physiological conditions. This has made it difficult to differentiate the individual roles of beta and gamma subunits in signal transduction. To characterize the individual subunits, the 36-kDa beta subunit (beta 1), brain gamma (gamma 2), and transducin gamma (gamma t) were translated in vitro in a rabbit reticulocyte lysate system. Hydrodynamic studies and tryptic proteolysis were used to compare the physical properties of the in vitro translation products with those of beta gamma dimers purified from bovine brain. The hydrodynamic studies indicate that, without gamma subunits, the beta subunits are not stable but tend to aggregate into high molecular weight complexes. When beta and gamma subunits were co-translated, stable beta gamma dimers formed that bound alpha 0 in a guanine nucleotide-dependent manner. The beta gamma dimers were less hydrophobic than those purified from bovine brain. This may reflect a lack of post-translational modification in the reticulocyte lysate or other differences between the in vitro translation products and the purified beta gamma. When beta and gamma were translated separately and then mixed, beta gamma dimers also formed. Analysis of in vitro translated beta gamma subunits will provide ways to assess the function of these subunits and to determine the structural requirements for beta gamma formation.
Schmidt
C J
CJ
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts.
Neer
E J
EJ
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014357:Trypsin; D019204:GTP-Binding Proteins