G beta gamma counteracts G alpha(q) signaling upon alpha(1)-adrenergic receptor stimulation

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.     

G beta association and effector interaction selectivities of the divergent G gamma subunit G gamma(13).

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.     

A universal allosteric mechanism for G protein activation

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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.     

Nucleotide sequence of the Belgian G gamma+(A gamma delta beta)0-thalassemia deletion breakpoint suggests a common mechanism for a number of such recombination events

Various types of thalassemia or hereditary persistence of fetal hemoglobin (HPFH) are caused by deletions at the human beta-globin gene cluster. Many of these molecular lesions show a clear clustering as far as size and location of their breakpoints are concerned. This might indicate common recombination mechanisms responsible for the generation of these deletions. The Belgian G gamma+(A gamma delta beta)zero-thalassemia results from a large deletion spanning the beta-globin gene cluster 3' of the A gamma gene. The extent of this deletion, analyzed by field-inversion gel electrophoresis, is approximately 50 kb and is very similar to that of the Indian HPFH (G gamma A gamma HPFH III) previously characterized by P. S. Henthorn et al. (1986). Proc. Natl. Acad. Sci. USA 83: 5194-5198. Isolation of the deletion junction of the Belgian G gamma+(A gamma delta beta)zero-thalassemia by means of inverse polymerase chain reaction confirmed a very close relationship between these two independent deletions. The 3' breakpoint of the Belgian deletion is located at the midpoint of a 160-bp palindrome, only four nucleotides 5' from the correspondent endpoint of the Indian HPFH.     

The role of G beta gamma and domain interfaces in the activation of G protein-coupled receptor kinase 2

In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on Galpha subunits, enabling both activated Galpha and Gbetagamma subunits to target downstream effector enzymes. One target of Gbetagamma is G protein-coupled receptor kinase 2 (GRK2), an enzyme that initiates homologous desensitization by phosphorylating activated GPCRs. GRK2 consists of three distinct domains: an RGS homology (RH) domain, a protein kinase domain, and a pleckstrin homology (PH) domain, through which it binds Gbetagamma. The crystal structure of the GRK2-Gbetagamma complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by Gbetagamma. In this paper, we report the 4.5 A structure of GRK2, which shows that the binding of Gbetagamma does not induce large domain rearrangements in GRK2, although small rotations of the RH and PH domains relative to the kinase domain are evident. Mutation of residues within the larger domain interfaces of GRK2 generally leads to diminished expression and activity, suggesting that these interfaces are important for stability and remain intact upon activation of GRK2. Geranylgeranylated Gbetagamma, but not a soluble mutant of Gbetagamma, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 A away from the Gbetagamma binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligomerization can account for the observed protection. The Gbetagamma-mediated binding of GRK2 to CHAPS micelles or lipid bilayers therefore appears to rigidify the kinase domain, perhaps by encouraging stable contacts between the RH and kinase domains.     

Molecular mechanism of the Syk activation switch

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.     

The role of beta2-glycoprotein I (beta2GPI) in the activation of plasminogen

Beta2-glycoprotein I (beta2GPI) is a glycoprotein of unknown physiological function. It is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome (APS). beta2GPI binds with high affinity to the atherogenic lipoprotein Lp(a) which shares structural homology with plasminogen, a key molecule in the fibrinolytic system. Impaired fibrinolysis has been described in APS. The present work reports the interaction between beta2GPI and Glu-Plasminogen which may explain the recently described proteolytic effect of plasmin on beta2GPI. In the process of Glu-Plasminogen activation, we found an increase in plasmin generation both at fibrin and cellular surface level as a function of the concentration of beta2GPI added, suggesting an important role as a cofactor in the trimolecular complex beta2GPI-Plasminogen-tPA. This phenomenon represents a novel regulatory step both in the positive feedback mechanism for extrinsic fibrinolysis and in antithrombotic regulation. IgG anti-beta2GPI antibodies recognized the beta2GPI at the endothelial surface inducing its activation with an increase of ICAM-I and a decrease in the expression of thrombomodulin favoring a pro-thrombotic state in the vascular endothelium. The interference in the plasmin conversion by anti-beta2GPI antibodies could generate thrombosis as observed in APS.     

The mating-specific G(alpha) protein of Saccharomyces cerevisiae downregulates the mating signal by a mechanism that is dependent on pheromone and independent of G(beta)(gamma) sequestration.

It has been inferred from compelling genetic evidence that the pheromone-responsive G(alpha) protein of Saccharomyces cerevisiae, Gpa1, directly inhibits the mating signal by binding to its own beta(gamma) subunit. Gpa1 has also been implicated in a distinct but as yet uncharacterized negative regulatory mechanism. We have used three mutant alleles of GPA1, each of which confers resistance to otherwise lethal doses of pheromone, to explore this possibility. Our results indicate that although the G322E allele of GPA1 completely blocks the pheromone response, the E364K allele promotes recovery from pheromone treatment rather than insensitivity to it. This observation suggests that Gpa1, like other G(alpha) proteins, interacts with an effector molecule and stimulates a positive signal--in this case, an adaptive signal. Moreover, the Gpa1-mediated adaptive signal is itself induced by pheromone, is delayed relative to the mating signal, and does not involve sequestration of G(beta)(gamma). The behavior of N388D, a mutant form of Gpa1 predicted to be activated, strongly supports these conclusions. Although N388D cannot sequester beta(gamma), as evidenced by two-hybrid analysis and its inability to complement a Gpa1 null allele under normal growth conditions, it can stimulate adaptation and rescue a gpa1(delta) strain when cells are exposed to pheromone. Considered as a whole, our data suggest that the pheromone-responsive heterotrimeric G protein of S. cerevisiae has a self-regulatory signaling function. Upon activation, the heterotrimer dissociates into its two subunits, one of which stimulates the pheromone response, while the other slowly induces a negative regulatory mechanism that ultimately shuts off the mating signal downstream of the receptor.     

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.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Restriction endonuclease mapping of gamma-delta-beta-globin region in G gamma (beta)+ HPFH and a Chinese A gamma HPFH variant.

Restriction endonuclease mapping of the beta-globin genomic region was used for studying the molecular basis of two variants of hereditary persistence of fetal hemoglobin (HPFH): an African G gamma (beta)+ HPFH and a Chinese HPFH variant with predominant synthesis of A gamma chains. HPFH and control DNA samples were digested with a battery of restriction enzymes, and the fragments were identified by hybridization to a family of discrete probes. DNA fragments from the A gamma HPFH (Chinese) and the G gamma (beta)+ HPFH individuals were identical with those of the normal controls. These findings suggest that the two mutants are the result of small structural anomalies of DNA sequences that play a role in the regulation of the expression of gamma-globin genes.     

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.     

Antidepressant-induced switch of beta 1-adrenoceptor trafficking as a mechanism for drug action

Reduction in surface beta(1)-adrenoceptor (beta1AR) density is thought to play a critical role in mediating the therapeutic long term effects of antidepressants. Since antidepressants are neither agonists nor antagonists for G protein-coupled receptors, receptor density must be regulated through processes independent of direct receptor activation. Endocytosis and recycling of the beta1AR fused to green fluorescent protein at its carboxyl-terminus (beta1AR-GFP) were analyzed by confocal fluorescence microscopy of live cells and complementary ligand binding studies. In stably transfected C6 glioblastoma cells, beta1AR-GFP displayed identical ligand-binding isotherms and adenylyl cyclase activation as native beta1AR. Upon exposure to isoproterenol, a fraction of beta1AR-GFP (10-15%) internalized rapidly and colocalized with endocytosed transferrin receptors in an early endosomal compartment in the perinuclear region. Chronic treatment with the tricyclic antidepressant desipramine (DMI) did not affect internalization characteristics of beta1AR-GFP when challenged with isoproterenol. However, internalized receptors were not able to recycle back to the cell surface in DMI-treated cells, whereas recycling of transferrin receptors was not affected. Endocytosed receptors were absent from structures that stained with fluorescently labeled dextran, and inhibition of lysosomal protease activity did not restore receptor recycling, indicating that beta1AR-GFP did not immediately enter the lysosomal compartment. The data suggest a new mode of drug action causing a "switch" of receptor fate from a fast recycling pathway to a slowly exchanging perinuclear compartment. Antidepressant-induced reduction of receptor surface expression may thus be caused by modulation of receptor trafficking routes.     

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma.

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).