Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric proteins that transduce extracellular signals into intracellular changes. Functionally different G proteins have been identified by their different a subunits. The 13 and ysubunits have been assumed to constitute a common pool shared among various G protein heterotrimers.

Guanine nucleotide-binding regulatory protein (G protein) by dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf`9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein .31 gene or a bovine G-protein 72 gene. It was possible to express the .i1 and 72 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant fi and/or y chains to function in dermed biochemical assays of fy activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing fi or y chain alone were inactive in these assays, whereas those from cells coinfected with fil and 72 did display activity. These assays were used to identify recombinant By dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble By dimers facilitated rhodopsin-catalyzed guanosine 5'-[ythioltriphosphate binding to Gta, the GTP-binding subunit of the retinal G protein transducin (KO.5 of 13 ± 2 and 36 ± 5 nM, respectively). Both recombinant fBy dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gta with equal potency (Ko.5 of 9 ± 1 and 10 ± 3 nM for membrane and soluble dimers, respectively). [3HIMevalono-lactone labeling showed that the 72 subunits of membraneassociated fBy dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of 72 directs the membrane association of the fi172 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.Guanine nucleotide-binding regulatory proteins (G proteins) are a family of proteins that serve as vital links in the chain of proteins that transduce extracellular signals to diverse intracellular effectors, such as adenylyl cyclases, phosphodiesterases, and phospholipases, and to ion channels in the membrane (1-3). All signal-transducing G proteins identified to date are membrane-associated heterotrimers composed of subunits referred to as a, 3, and 'y. The (3 and y subunits of G proteins do not dissociate under physiological conditions and thus behave essentially as a monomer; in addition, fBy dimers occur in almost all tissues as a complex mixture oftwo 3proteins (4), each bound to as many as three y subunits (5, 6). Gf3'y displays a number of in vitro activities related to the guanine nucleotide binding and activation of a subunits, including reversal of aluminum fluoride-activated Gsa (ref. 7; G., stimulatory G protein), inhibition or reversal ofguanosine 5'-[y-thio]triphosphate (GTP[yS]) binding to Ga (8,9), and enhancement of pertussis and cholera toxin catalyzed ADPribosylation of Ga subunits (10, 11). Gfy is required for efficient coupling of Ga subunits to cell surface receptors (12-15). Fina...

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 Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

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