Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Specificity of G protein beta and gamma subunit interactions.

Multiple heterotrimeric guanine nucleotide binding protein (G protein) subunits have evolved to couple a large variety of receptors to intracellular effectors. G protein beta gamma subunits are essential for efficient coupling of alpha subunits to receptors, and they are also important for modulation of effectors. Several different beta and gamma subunits exist, but it is not known whether all possible combinations of beta and gamma can form functional dimers. To answer this question, we have compared the ability of in vitro translated beta 1, beta 2, and beta 3 to form dimers with either gamma 1 or gamma 2. Dimerization was monitored by gel filtration, resistance to tryptic digestion, and chemical cross-linking. The results indicate that beta 1 binds both gamma subunits, beta 2 binds only gamma 2, and beta 3 will bind neither gamma 1 or gamma 2. Hence, the occurrence of beta gamma dimers may be partially regulated by the ability of the subunits to associate. Specificity of dimerization might allow cells to co-express multiple beta and gamma subunits while maintaining efficient and specific signal transduction.     

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.     

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.     

Specificity and structural requirements of phospholipase C-beta stimulation by Rho GTPases versus G protein beta gamma dimers

Phospholipase C-beta(2) (PLC beta(2)) is activated both by heterotrimeric G protein alpha- and beta gamma- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC beta isozymes with the rank order of PLC beta(2) > PLC beta(3) > or = PLC beta(1). The sensitivity of PLC beta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLC beta(3) > PLC beta(2) > PLC beta(1) for beta(1)gamma(1/2) and PLC beta(2) > PLC beta(1) > PLC beta(3) for beta(5)gamma(2). Rac1 and Rac2 were found to be more potent and efficacious activators of PLC beta(2) than was Cdc42Hs. The stimulation of PLC beta(2) by Rho GTPases and G protein beta gamma dimers was additive, suggesting that PLC beta(2) activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC beta(1)-PLC beta(2) enzymes, beta gamma dimers, and Rho GTPases are shown to require different regions of PLC beta(2) to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC beta(2) were sufficient for efficient stimulation by beta gamma, the presence of the putative pleckstrin homology domain of PLC beta(2) was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC beta regulators, which mediate PLC beta isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC beta(2) by extracellular mediators in intact cells.     

Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers

The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.     

Differential sensitivity of P-Rex1 to isoforms of G protein betagamma dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.     

Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit in determining effector activation.

Post-translational prenylation of heterotrimeric G protein gamma subunits is essential for high affinity alpha-beta gamma and alpha-beta gamma-receptor interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in the interaction of beta gamma dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the gamma1, gamma2, and gamma11 subunits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase. The native beta1 gamma2 dimer (gamma subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-beta than either the beta1 gamma1 (farnesyl) or the beta1 gamma11 (farnesyl) dimers. However, farnesyl modification of the gamma subunit in the beta1 gamma2 dimer (beta1 gamma2-L71S) caused a decrement in its ability to activate phospholipase C-beta. In contrast, both the beta1 gamma1-S74L (geranylgeranyl) and the beta1 gamma11-S73L (geranylgeranyl) dimers were more active than the native forms. The beta1 gamma2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta1 gamma1 nor the beta1 gamma11 dimers activate the enzyme. As was the case with phospholipase C-beta, the beta1gamma2-L71S dimer was less able to activate adenylyl cyclase than the native beta1 gamma2 dimer. Interestingly, neither the beta1 gamma1-S74L nor the beta1 gamma11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the gamma subunit and its prenyl group play a role in determining the activity of the beta gamma-effector complex.     

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.     

Characterization of heterotrimeric G protein complexes in rice plasma membrane

Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells. Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit. When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

Ribozyme approach identifies a functional association between the G protein beta1gamma7 subunits in the beta-adrenergic receptor signaling pathway.

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effectors. However, in most cases, the specific combinations of alpha and betagamma subunits comprising the G proteins that participate in the most common signaling pathways, such as beta-adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the betagamma subunits that combine with the alpha subunit of Gs are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the gamma7 subunit of the G proteins, thereby identifying a specific role of this protein in coupling the beta-adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells. In the present study, we explored the potential utility of a ribozyme approach directed against the gamma7 subunit to identify functional associations with a particular beta and alphas subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the gamma7 subunit, and the effects of this manipulation on levels of the beta and alphas subunits were determined by immunoblot analysis. Among the five beta alphas subunits detected in these cells, only the beta1 subunit was coordinately reduced following treatment with the ribozyme directed against the gamma7 subunit, thereby demonstrating a functional association between the beta1 and gamma7 subunits. The mechanism for coordinate suppression of the beta1 subunit was due to a striking change in the half-life of the beta1 monomer versus the beta1 heterodimer complexed with the gamma7 subunit. Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme directed against the gamma7 subunit, thereby providing insights into the assembly of the Gs heterotrimer. Taken together, these data show the utility of a ribozyme approach to identify the role of not only the gamma subunits but also the beta subunits of the G proteins in signaling pathways.     

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective G protein beta gamma-subunit compositions mediate phospholipase C activation in the vomeronasal organ

Chemosensory neurons of the vomeronasal organ (VNO) are supposed to detect pheromones controlling social and reproductive behavior in most terrestrial vertebrates. Recent studies indicate that pheromone signaling in VNO neurons is mediated via phospholipase C (PLC) activation generating the two second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Since G alpha(i) and G alpha(o) predominantly expressed in VNO neurons are usually not involved in activating PLC, it was explored if PLC activation may be mediated by G beta gamma subunits. It was found that a scavenger for beta gamma dimers reduced the urine-induced IP3 formation in VNO preparations in a dose-dependent manner indicating a role for G beta gamma complexes. Towards an identification of the relevant G beta and G gamma subunit(s), PCR approaches as well as immunohistochemical experiments were performed. It was found that out of the five known G beta subtypes, only G beta2 was expressed in both G alpha(i) as well as G alpha(o) neurons. Experimental approaches focusing on the spatial expression profile of identified G gamma subtypes revealed that G gamma8-positive neurons are preferentially localized to the basal region of the vomeronasal epithelium, whereas G gamma2-reactive cells are restricted to the apical G alpha(i)-positive layer of the sensory epithelium. As IP3 formation induced upon stimulation with volatile urinary compounds was selectively blocked by G gamma2-specific antibodies whereas second messenger formation elicited upon stimulation with alpha2u globulin was inhibited by antibodies recognizing G gamma8, it is conceivable that PLC activation in the two populations of chemosensory VNO neurons is mediated by different G beta gamma complexes.     

Selective association of G protein beta(4) with gamma(5) and gamma(12) subunits in bovine tissues.

The beta and gamma subunits of G proteins are tightly bound under physiological conditions, and so far, seven beta and 11 gamma subunit isoforms have been found. The relative abilities of the beta and gamma subunits to associate with each other have been studied using transfected cell assays, in vitro translation and the yeast two-hybrid system, but have not been fully characterized in various tissues. In the present study, we demonstrated the selectivity of association of the beta with gamma isoforms in bovine tissues. Immunoprecipitation of betagamma complexes from tissue extracts with antibodies against various gamma subunits and subsequent analyses revealed that beta(4) associated with the gamma subunits with the following rank order of selectivity: gamma(5) > gamma(12) > gamma(2) > gamma(3), while beta(2) bound to gamma(2), gamma(3), and gamma(12) more selectively than to gamma(5). By contrast, beta(1) associated with all gamma subunits without significant selectivity. Analyses of purified betagamma complexes containing various gamma isoforms revealed beta subunit compositions similar to those found in the immunoprecipitates. Particular combinations of beta and gamma subunit isoforms may contribute to maintaining efficient and specific signal transduction mediated by G proteins.     

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.     

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.     

Selective role of G protein gamma subunits in receptor interaction

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy. However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5. Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai. These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.     

Chromosomal mapping of five mouse G protein gamma subunits

Heterotrimeric G proteins, composed of alpha, beta, and gamma subunits, transduce signals from transmembrane receptors to a wide range of intracellular effectors. The G protein gamma subunits, which play an indispensible role in this communication, constitute a large and diverse multigene family. Using an interspecific backcross panel, we have determined the mouse chromosomal locations of five gamma subunit genes: gamma2, gamma8, gamma10, gamma12, and gammaCone. Combined with previous mapping studies, these data indicate that, with the possible exception of gamma1 and gamma11, the G protein gamma subunit genes are well dispersed within the mouse and human genomes.     

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.