Gbetagamma Dimers Research Articles

In vivo expression of G‐protein β1γ2 dimer in adult mouse skeletal muscle alters L‐type calcium current and excitation–contraction coupling

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Stargazin Modulates Neuronal Voltage-dependent Ca2+ Channel Cav2.2 by a Gβγ-dependent Mechanism

Loss of neuronal protein stargazin (gamma(2)) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel gamma(1) subunit, stargazin and other family members (gamma(3-8)) were classified as gamma subunits of neuronal voltage-gated calcium channels (such as Ca(V)2.1-Ca(V)2.3). Here, we report that stargazin interferes with G protein modulation of Ca(V)2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gbetagamma-induced inhibition of Ca(V)2.2 channel currents, caused either by coexpression of the Gbetagamma dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gbetagamma overcame the effects of stargazin. High affinity Gbetagamma scavenger proteins m-cbetaARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cbetaARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the Ca(V)beta(3) subunit. However, in some cases, coexpression of Ca(V)beta(3) blunted the modulation by stargazin. Finally, the Gbetagamma-opposing action of stargazin was not unique to Ca(V)2.2, as stargazin also inhibited the Gbetagamma-mediated activation of the G protein-activated K(+) channel. Purified cytosolic C-terminal part of stargazin bound Gbetagamma in vitro. Our results suggest that the regulation by stargazin of biophysical properties of Ca(V)2.2 are not exerted by direct modulation of the channel but via a Gbetagamma-dependent mechanism.

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

Fonction des chaperonnes moléculaires dans l’assemblage des protéines G hétérotrimériques

Extracellular signals received by G protein-coupled receptors (GPCRs) are transduced into intracellular responses following the activation of heterotrimeric G proteins. As their names suggests, they are composed of three subunits, Galpha and Gbetagamma, the latter being effectively treated as a single entity. The Gbetagamma dimer is assembled with the aid of a number of molecular chaperones in a tightly regulated process. The folding of nascent Gbeta1 is favoured by cellular chaperones such as PhLP-1 and CCT and the ER-resident protein DRiP78 plays an important role in the stability of nascent Ggamma2 subunits. However, much work remains to be done to completely understand the mechanisms underlying assembly of the heterotrimer.

Blockade of Ethanol-Induced Potentiation of Glycine Receptors by a Peptide That Interferes with Gβγ Binding

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gbetagamma. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gbetagamma to 21 +/- 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gbetagamma binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gbetagamma dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gbetagamma to GlyRs to 57 +/- 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gbetagamma-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 +/- 14% of control) and attenuated the GlyR potentiation by ethanol (51 +/- 10% versus 10 +/- 3%).

Naloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor–Gs Coupling and CREB Activation of Acute Morphine

Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Gαs and its Gβγ dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 µM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA2561–2565), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR–FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR–Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR–Gs coupling by 100 pM NLX/NTX or 10 µM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.

A Point Mutation to Gαi Selectively Blocks GoLoco Motif Binding: DIRECT EVIDENCE FOR Gα·GoLoco COMPLEXES IN MITOTIC SPINDLE DYNAMICS

Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.

RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2

A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta.

Activation of Human Phospholipase C-η2 by Gβγ

Phospholipase C-eta2 (PLC-eta2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta2 by Gbetagamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta2 with Gbeta 1gamma 2, as well as with certain other Gbetagamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gbeta 1gamma 2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-eta2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta2 and its potential direct activation by Gbetagamma, a construct of PLC-eta2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta2 was quantified after reconstitution with PtdIns(4,5)P 2-containing phospholipid vesicles, and values for K m (14.4 microM) and V max [12.6 micromol min (-1) (mg of protein) (-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified Gbeta 1gamma 2 stimulated the activity of purified PLC-eta2 in a concentration-dependent manner similar to that observed with purified PLC-beta2. Activation was dependent on the presence of free Gbeta 1gamma 2 since its sequestration in the presence of Galpha i1 or GRK2-ct reversed Gbeta 1gamma 2-promoted activation. The PH domain of PLC-eta2 is not required for Gbeta 1gamma 2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gbeta 1gamma 2. Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and, therefore, of receptor-activated heterotrimeric G proteins.

Gβγ Dimers Released in Response to Thyrotropin Activate Phosphoinositide 3-Kinase and Regulate Gene Expression in Thyroid Cells

Signaling by TSH through its receptor leads to the dissociation of trimeric G proteins into Galpha and Gbetagamma. Galphas activates adenylyl cyclase, which increases cAMP levels that induce several effects in the thyroid cell, including transcription of the sodium-iodide symporter (NIS) gene through a mechanism involving Pax8 binding to the NIS promoter. Much less is known about the function of Gbetagamma in thyroid differentiation, and therefore we studied their role in TSH signaling. Gbetagamma overexpression inhibits NIS promoter activation and reduces NIS protein accumulation in response to TSH and forskolin. Conversely, inhibition of Gbetagamma-dependent pathways increases NIS promoter activity elicited by TSH but does not modify forskolin-induced activation. Gbetagamma dimers are being released from the Gs subfamily of proteins, because cholera toxin mimics the effects elicited by TSH, whereas pertussis toxin has no effect on NIS promoter activity. We also found that TSH stimulates Akt phosphorylation in a phosphoinositide 3-kinase (PI3K)-dependent and cAMP-independent manner. This is mediated by Gbetagamma, because its overexpression or specific sequestration, respectively, increased or reduced phosphorylated Akt levels upon TSH stimulation. Gbetagamma sequestration increases NIS protein levels induced by TSH and Pax8 binding to the NIS promoter, which is also increased by PI3K inhibition. This is, at least in part, caused by Gbetagamma-mediated Pax8 exclusion from the nucleus that is attenuated when PI3K activity is blocked. These data unequivocally demonstrate that Gbetagamma released by TSH action stimulate PI3K, inhibiting NIS gene expression in a cAMP-independent manner due to a decrease in Pax8 binding to the NIS promoter.

Heterotrimeric G protein gamma subunits provide functional selectivity in Gbetagamma dimer signaling in Arabidopsis.

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

The G‐protein coupling properties of the human sweet and amino acid taste receptors

The human T1R taste receptors are family C G-protein-coupled receptors (GPCRs) that act as heterodimers to mediate sweet (hT1R2 + hT1R3) and umami (hT1R1 + hT1R3) taste modalities. Each T1R has a large extracellular ligand-binding domain linked to a seven transmembrane-spanning core domain (7TMD). We demonstrate that the 7TMDs of hT1R1 and hT1R2 display robust ligand-independent constitutive activity, efficiently catalyzing the exchange of GDP for GTP on Galpha subunits. In contrast, relative to the 7TMDs of hT1R1 and hT1R2, the 7TMD of hT1R3 couples poorly to G-proteins, suggesting that in vivo signaling may proceed primarily through hT1R1 and hT1R2. In addition, we provide direct evidence that the hT1Rs selectively signal through Galpha(i/o) pathways, coupling to multiple Galpha(i/o) subunits as well as the taste cell specific Gbeta(1)gamma(13) dimer.

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

It is generally accepted that G protein coupled receptors (GPCR) activate heterotrimeric G proteins by inducing a GDP/GTP exchange at the G protein alpha subunit. In addition, the transfer of high energetic phosphate by nucleoside diphosphate kinase (NDPK) and/or the beta subunit of G proteins (Gbeta) can induce G protein activation. Recent evidence suggests that the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers. In this complex, NDPK B acts as a protein histidine kinase phosphorylating Gbeta at histidine residue 266 (His266). The high energetic phosphoamidate bond on His266 allows for a phosphate transfer specifically onto GDP and thus local formation of GTP, which binds to and thereby activates the respective G protein alpha subunit. Apparently, this process occurs independent of the classical GPCR-induced GDP/GTP exchange at least for members of the G(s) and G(i) subfamilies of heterotrimeric G proteins. By using a mutant of Gbeta(1) in which His266 was replaced by Leu, it was recently demonstrated that NDPK B/Gbetagamma-mediated G(s) activation contributes by about 50% to basal cAMP formation and contractility in rat cardiac myocytes. Besides its apparent role in G protein activation, the complex formation of NDPK B with Gbetagamma dimers might be essential for G protein stability. Depletion of either the NDPK B orthologue or Gbeta(1) isoforms in zebrafish embryos led to a similar phenotype displaying contractile dysfunction in the heart accompanied by a complete loss of heterotrimeric G protein expression. In conclusion, the interaction of NDKP B with Gbetagamma dimers might play an important role in signal transduction, and alterations in this novel pathway might be of pathophysiological importance.

High energy phosphate transfer by NDPK B/Gβγcomplexes - an alternative signaling pathway involved in the regulation of basal cAMP production

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein alpha -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the beta-subunit of G proteins (Gbeta) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers in which NDPK B acts as a histidine kinase phosphorylating Gbeta at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein alpha-subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.

Dual Role for Membrane Localization in Yeast MAP Kinase Cascade Activation and Its Contribution to Signaling Fidelity

Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.

A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

Activation of mitogen-activated protein (MAP) kinase cascade signaling by yeast mating pheromones involves recruitment of the Ste5 scaffold protein to the plasma membrane by the receptor-activated Gbetagamma dimer. Here, we identify a putative amphipathic alpha-helical domain in Ste5 that binds directly to phospholipid membranes and is required for membrane recruitment by Gbetagamma. Thus, Ste5 signaling requires synergistic Ste5-Gbetagamma and Ste5-membrane interactions, with neither alone being sufficient. Remarkably, the Ste5 membrane binding domain is a dual-function motif that also mediates nuclear import. Separation-of-function mutations show that signaling requires the membrane-targeting activity of this domain, not its nuclear-targeting activity, and heterologous lipid binding domains can substitute for its function. This domain also contains imperfections that reduce membrane affinity, and their elimination results in constitutive signaling, explaining some previous hyperactive Ste5 mutants. Therefore, weak membrane affinity is advantageous, ensuring a normal level of signaling quiescence in the absence of stimulus and imposing a requirement for Gbetagamma binding.

The Gβ(KlSte4p) subunit of the heterotrimeric G protein has a positive and essential role in the induction of mating in the yeast Kluyveromyces lactis

In the yeast Saccharomyces cerevisiae the Gbetagamma dimer of the heterotrimeric G protein transduces a pheromone signal from serpentine receptor to a MAP kinase cascade that activates the mating response pathway. Haploid cells lacking the Gbeta subunit do not respond to sexual pheromone, leading to sterility. In this work we demonstrate that the beta-subunit of Kluyveromyces lactis, encoded by the KlSTE4 gene, is a component of the G protein, and that its disruption gives rise to sterile cells. However, unlike Ste4p in S. cerevisiae, its overexpression does not induce growth arrest or promote mating. It has been shown that in K. lactis, the Galpha subunit has a positive role in the mating process, hence the resulting double GalphaDelta GbetaDelta mutant was viable and sterile. Here we show that the overproduction of Gbeta subunit fails to rescue GalphaDelta mutant from sterility and that expression of a constitutive active allele of Galpha enhances transcription of the KlSTE4 gene. The mating pathway triggered by the Gbeta-subunit requires a functional KlSte12p transcription factor. Gbeta has a 10-fold higher association rate with the Galpha1 subunit involved in pheromone response than with Galpha2, the protein involved in cAMP regulation in K. lactis. Additionally, the Gbeta-subunit from K. lactis is able to interact with the Galpha-subunit from S. cerevisiae but fails to restore the mating deficiency of Scste4Delta mutant. The data presented indicate that the mating pathway of K. lactis is positively and cooperatively regulated by both the Galpha and the Gbeta subunits.

G Protein βγ Dimer Formation: Gβ and Gγ Differentially Determine Efficiency of in Vitro Dimer Formation

The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.

Influence of the Membrane Lipid Structure on Signal Processing via G Protein-Coupled Receptors

We have recently reported that lipid structure regulates the interaction with membranes, recruitment to membranes, and distribution to membrane domains of heterotrimeric Galphabetagamma proteins, Galpha subunits, and Gbetagamma dimers (J Biol Chem 279:36540-36545, 2004). Here, we demonstrate that modulation of the membrane structure not only determines G protein localization but also regulates the function of G proteins and related signaling proteins. In this context, the antitumor drug daunorubicin (daunomycin) and oleic acid changed the membrane structure and inhibited G protein activity in biological membranes. They also induced marked changes in the activity of the alpha(2A/D)-adrenergic receptor and adenylyl cyclase. In contrast, elaidic and stearic acid did not change the activity of the above-mentioned proteins. These fatty acids are chemical but not structural analogs of oleic acid, supporting the structural basis of the modulation of membrane lipid organization and subsequent regulation of G protein-coupled receptor signaling. In addition, oleic acid (and also daunorubicin) did not alter G protein activity in a membrane-free system, further demonstrating the involvement of membrane structure in this signal modulation. The present work also unravels in part the molecular bases involved in the antihypertensive (Hypertension 43:249-254, 2004) and anticancer (Mol Pharmacol 67:531-540, 2005) activities of synthetic oleic acid derivatives (e.g., 2-hydroxyoleic acid) as well as the molecular bases of the effects of diet fats on human health.

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.