Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha-subunits in association with a tightly bound complex of beta and gamma-subunits. While the alpha-subunits are recognized as a family of diverse structures, beta and gamma-subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta-gamma-complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma-subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta-isoforms (beta-36 and beta-35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta-36 and two for beta-35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta-gamma-complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured-beta. In contrast to the two highly homologous beta-structures, examination of this set of proteins revealed at least six distinct gamma-peptides. Two unique gamma-peptides were found in bovine retinal G(t) and three gamma-peptides in samples of bovine brain derived G(o)/G(i). Human placental and platelet G(i) samples each contained a unique-gamma. Finally, rabbit liver G(i) preparations contained three electrophoretically resolvable gamma-peptides. Antisera raised to the retinal beta-gamma structure recognize both retinal gamma-forms and no other forms. The larger gamma-peptide from liver G(i) is recognized by antiserum beta-8 which specifically recognizes the human placental-gamma, the most rapidly migrating-gamma from liver corresponds with the mobility of the human platelet-gamma, and the third liver-gamma was found in our previous study to be recognized by a sequence-specific antiserum which identifies one of the two closely migrating G(o)/G(i)-gamma's as the product of the gamma-2 gene [Gautam, N., Northup, J. K., Tamir, H., & Simon, M. I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7973-7977]. An additional gamma-peptide found in some, but not all, G(o)/G(i) preparations was identified as gamma-3. These studies show a far greater diversity of the gamma-subunit structure than had previously been recognized. Our results suggest that the function of the beta-subunit in G-proteins is highly conserved and that biochemical differences among the G-beta-gamma forms are likely to be due to the diversity in gamma-structures.
