The G protein beta subunit is a determinant in the coupling of Gs to the beta 1-adrenergic and A2a adenosine receptors.

The signaling specificity of five purified G protein betagamma dimers, beta(1)gamma(2), beta(2)gamma(2), beta(3)gamma(2), beta(4)gamma(2), and beta(5)gamma(2), was explored by reconstituting them with G(s) alpha and receptors or effectors in the adenylyl cyclase cascade. The ability of the five betagamma dimers to support receptor-alpha-betagamma interactions was examined using membranes expressing the beta(1)-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta isoform. The beta(4)gamma(2) dimer demonstrated the highest coupling efficiency to either receptor. The beta(5)gamma(2) dimer coupled poorly to each receptor, with EC(50) values 40-200-fold higher than those observed with beta(4)gamma(2). Strikingly, whereas the EC(50) of the beta(1)gamma(2) dimer at the beta(1)-adrenergic receptor was similar to beta(4)gamma(2), its EC(50) was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the betagamma dimers were measured. betagamma dimers containing Gbeta(1-4) were able to stimulate AC2 similarly, and beta(5)gamma(2) was much less potent. beta(1)gamma(2), beta(2)gamma(2), and beta(4)gamma(2) inhibited AC1 equally; beta(3)gamma(2) was 10-fold less effective, and beta(5)gamma(2) had no effect. These data argue that the beta isoform in the betagamma dimer can determine the specificity of signaling at both receptors and effectors.

Activation and inhibition of G protein-coupled inwardly rectifying potassium (Kir3) channels by G protein beta gamma subunits.

G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein betagamma subunits containing either beta1 or beta2 with multiple Ggamma subunits activate GIRK channels, we hypothesized that specificity might be imparted by beta3, beta4, or beta5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these Gbeta subunits. Inwardly rectifying K(+) currents were increased in cells expressing beta3 or beta4, with either gamma2 or gamma11. Purified, recombinant beta3gamma2 and beta4gamma2 bound directly to glutathione-S-transferase fusion proteins containing N- or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that beta3 and beta4, like beta1, form dimers that bind to and activate GIRK channels. By contrast, beta5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either beta5gamma2 or beta5gamma11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous Gbetagamma subunits. In cotransfection assays, beta5gamma2 suppressed beta1gamma2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that beta5gamma2 could bind to the same GIRK channel cytoplasmic domains as other, activating Gbetagamma subunits. Thus, beta5-containing dimers inhibit Gbetagamma-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that beta5-containing dimers could act as competitive antagonists of other Gbetagamma dimers on GIRK channels.

Selective coupling of G protein beta gamma complexes to inhibition of Ca2+ channels.

Several mechanisms couple heterotrimeric guanine nucleotide-binding proteins (G proteins) to cellular effectors. Although alpha subunits of G proteins (Galpha) were the first recognized mediators of receptor-effector coupling, Gbetagamma regulation of effectors is now well known. Five Gbeta and 12 Ggamma subunit genes have been identified, suggesting through their diversity that specific subunits couple selectively to effectors. The molecular determinants of Gbetagamma-effector coupling, however, are not well understood, and most studies of G protein-effector coupling do not support selectivity of Gbetagamma action. To explore this issue further, we have introduced recombinant Gbetagamma complexes into avian sensory neurons and measured the inhibition of Ca(2+) currents mediated by an endogenous phospholipase Cbeta- (PLCbeta) and protein kinase C-dependent pathway. Activities of Gbetagamma in the native cells were compared with enzyme assays performed in vitro. We report a surprising selective activation of the PLCbeta pathway by Gbetagamma complexes containing beta(1) subunits, whereas beta(2)-containing complexes produced no activation. In contrast, when assayed in vitro, PLCbeta and type II adenylyl cyclase did not discriminate among these same Gbetagamma complexes, suggesting the possibility that additional cellular determinants confer specificity in vivo.

The relationship of G(o)alpha subunit deamidation to the tissue distribution, nucleotide binding properties, and betagamma dimer interactions of G(o)alpha subunit isoforms.

The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differed substantially from that of alpha(o)A and alpha(o)C, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C showed slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurred in a constant ratio to alpha(o)A throughout the brain, but was a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain.

Characterization of the major bovine brain Go alpha isoforms. Mapping the structural differences between the alpha subunit isoforms identifies a variable region of the protein involved in receptor interactions.

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.

A major G protein alpha O isoform in bovine brain is deamidated at Asn346 and Asn347, residues involved in receptor coupling.

The structural differences between two major forms of the alpha subunit of the heterotrimeric G protein GO were found to be due to deamidation of either of two Asn residues near the C-terminus of the proteins, in a region involved in receptor recognition. GO is the most abundant heterotrimeric G protein in mammalian brain. Two forms of the protein, GOA and GOB, are known to be generated by alternative splicing of a single GOalpha gene. A third isoform, alphaOC, represents about 1/3 of the alphaO protein in brain and is related to alphaOA, from which it is thought to be generated by protein modification. Mass spectrometry and chemical derivatization of tryptic fragments of the proteins were used to localize the structural difference between alphaOA and alphaOC to a C-terminal peptide. Sequence analysis of a C-terminal chymotryptic fragment both by ion trap mass spectrometry and by Edman degradation identified Asn346 and Asn347 of alphaOA as alternative deamidation sites in alphaOC. These structural differences have immediate implications for G protein function, as they occur in a conformationally sensitive part of the protein involved in receptor recognition and activation. Since Asn347 is a conserved residue present in most G protein alpha subunits outside the alphas family, these observations may have general significance for many G proteins. Deamidation may be a component of a novel process for modifying or adapting cellular responses mediated by G proteins.

Bovine brain GO isoforms have distinct gamma subunit compositions.

The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.