Compartmentalization of protein kinase A signaling by the heterotrimeric G protein Go

G(o), a member of the G(o/i) family, is the most abundant heterotrimeric G protein in brain. Most functions of G(o) are mediated by the G(betagamma) dimer; effector(s) for its alpha-subunit have not been clearly defined. Here we report that G(oalpha) interacts directly with cAMP-dependent protein kinase (PKA) through its GTPase domain. This interaction did not inhibit the kinase function of PKA but interfered with nuclear translocation of PKA while sparing its cytosolic function. This regulatory mechanism by which G(o) bifurcates PKA signaling may provide insights into how G(o) regulates complex processes such as neuritogenesis, synaptic plasticity, and cell transformation.     

Regulation of heterotrimeric G protein signaling in airway smooth muscle

Heterotrimeric G proteins transduce signals from G protein-coupled receptors to regulate numerous signaling events and functions in airway smooth muscle (ASM). In this article, we detail the function and regulation of heterotrimeric G protein signaling in ASM. We further discuss recent advances in the development of experimental tools in the study of G protein signaling, and speculate how these tools might be used in therapeutic strategies that seek to mitigate bronchospasm and airway remodeling that occur in obstructive airway disease.     

Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit

Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes conformational changes in three regions of G alpha, including Switch I and Switch II. Mutation of G202-->A in Switch II of G alpha(i1) accelerates the rates of both GTP hydrolysis and conformational change, which is measured by the loss of fluorescence from Trp-211 in Switch II. Mutation of K180-->P in Switch I increases the rate of conformational change but decreases the GTPase rate, which causes transient but substantial accumulation of a low-fluorescence G alpha(i1).GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)G alpha(i1) and (K180P)G alpha(i1) to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of G alpha(i1) that is mimicked by the complex of G alpha(i1) with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)G alpha(i1) bound to a slowly hydrolyzable GTP analog, and the GDP.magnesium fluoroaluminate complex provide evidence that the Mg(2+) binding site is destabilized and that Switch I is torsionally restrained by the K180P mutation. The data are consistent with a catalytic mechanism for G alpha in which major conformational transitions in Switch I and Switch II are obligate events that precede the bond-breaking step in GTP hydrolysis. In (K180P)G alpha(i1), the two events are decoupled kinetically, whereas in the native protein they are concerted.     

Cardioprotection specific for the G protein Gi2 in chronic adrenergic signaling through beta 2-adrenoceptors

Two subtypes of beta-adrenoceptors, beta 1 and beta 2, mediate cardiac catecholamine effects. These two types differ qualitatively, e.g., regarding G protein coupling and calcium channel stimulation. Transgenic mice overexpressing human beta 2-adrenoceptors survive high-expression levels, unlike mice overexpressing beta 1-adrenoceptors. We examined the role of inhibitory Gi proteins, known to be activated by beta 2- but not beta 1-adrenoceptors, on the chronic effects of human beta 2-adrenoreceptor overexpression in transgenic mice. These mice were crossbred with mice where G alpha i2, a functionally important cardiac Gi alpha-subunit, was inactivated by targeted gene deletion. Survival of beta 2-adrenoreceptor transgenic mice was reduced by heterozygous inactivation of G alpha i2. Homozygous knockout/beta 2-adrenoreceptor transgenic mice died within 4 days after birth. Heterozygous knockout/beta 2-adrenoreceptor transgenic mice developed more pronounced cardiac hypertrophy and earlier heart failure compared with beta 2-adrenoreceptor transgenic mice. Single calcium-channel activity was strongly suppressed in heterozygous knockout/beta 2-adrenoreceptor transgenic mice. In cardiomyocytes from these mice, pertussis toxin treatment in vitro fully restored channel activity and enhanced channel activity in cells from homozygous G alpha i2 knockout animals. Cardiac G alpha i3 protein was increased in all G alpha i2 knockout mouse strains. Our results demonstrate that G alpha i2 takes an essential protective part in chronic signaling of overexpressed beta 2-adrenoceptors, leading to prolonged survival and delayed cardiac pathology. However, reduction of calcium-channel activity by beta 2-adrenoreceptor overexpression is due to a different pertussis-toxin-sensitive pathway, most likely by G alpha i3. This result indicates that subtype-specific signaling of beta 2-adrenoreceptor functionally bifurcates at the level of Gi, leading to different effects depending on the G alpha isoform.     

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.     

Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice

Transgenic rice containing an antisense cDNA for the alpha subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the alpha subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the alpha-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the alpha subunit by antisense technology. Analysis of the cDNAs for the alpha subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361-1, showed that these dwarf mutants had mutated in the coding region of the alpha-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice.     

The obligatory action of protein tyrosine phosphatases in ACTH-stimulated steroidogenesis is exerted at the level of StAR protein

A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 microM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 +/- 30, ACTH + PAO = 145 +/- 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.     

Structural and dynamical changes in an alpha-subunit of a heterotrimeric G protein along the activation pathway

The Galpha subunits of heterotrimeric G proteins (Galphabetagamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in "switch" sequences of Galpha, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gbetagamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Galphai1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix alpha4, a putative effector binding region. In the inactive Galphai1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gbetagamma, motions of R1 are highly constrained, reflecting direct contact interactions at the Galphai1-Gbeta interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding alpha2/beta4 loop and smaller allosteric changes at the Galphai1-Gbetagamma interface distant from the receptor binding surface. Upon addition of GTPgammaS, the activated Galphai1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in alpha4, distant from the interaction surface, supports a role for this helix in effector binding.     

The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.     

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.     

Association of the G protein beta3 subunit T allele with insulin resistance in essential hypertension

A polymorphism (C825T) in the gene encoding the G protein beta3 subunit (GNB3) has recently been associated with hypertension and obesity in several populations. The aim of the study was to analyse the relationship between this polymorphism and insulin sensitivity, an hypothesised unifying factor for hypertension and obesity. One hundred thirty unrelated patients with essential hypertension, 70 female and 60 male, aged 58 +/- 1 years with systolic blood pressure of 173 +/- 2 mm Hg and diastolic blood pressure of 105 +/- 1 mm Hg, were genotyped for the GNB3 polymorphism by PCR and restriction digestion with BseDI, and classified in two groups according to the genotypes CC and CT + TT. Body mass index (BMI) was significantly higher in patients with the T allele as compared with patients without the T allele (29.3 +/- 0.4 vs. 26.7 +/- 0.6 kg/m2, p<0.001). On the contrary, there were no differences in the level of systolic or diastolic blood pressure among the genotypes. Insulin sensitivity was measured in a subgroup of 35 patients by means of an euglycemic hyperinsulinemic clamp test. In this subgroup, patients with the T allele displayed lower insulin sensitivity index (1.6 +/- 0.3 vs. 2.7 +/- 0.3 mg/kg/min, p = 0.022), higher fasting serum insulin (121 +/- 16 vs. 77 +/- 11 pmol/L, p = 0.032), higher serum glucose 120 min after 75 g load (9.8 +/- 1.2 vs. 7.0 +/- 0.5 mmol/L, p = 0.038), and higher glycosilated haemoglobin (5.7 +/- 0.4 vs. 4.7 +/- 0.2%; p = 0.042) as compared with patients without the T allele. A regression analysis showed that the association between the T allele and insulin sensitivity was independent of BMI (beta coefficient -0.386, p = 0.022). These results suggest a relationship between the 825T allele of GNB3 and insulin resistance in the essential hypertensive patients studied, which seems to be independent of BMI.     

The heterotrimeric G protein alpha subunit acts upstream of the small GTPase Rac in disease resistance of rice

We used rice dwarf1 (d1) mutants lacking a single-copy Galpha gene and addressed Galpha's role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H(2)O(2) production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Galpha mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Galpha in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling.     

Glyburide but not ciglitazone enhances insulin action in the liver independent of insulin receptor kinase activation

To test the hypothesis that sulfonylureas enhance insulin action by activating the insulin receptor tyrosine kinase, the effects of glyburide, a second generation sulfonylurea, and ciglitazone, a nonsulfonylurea hypoglycemic agent, were determined in primary cultures of rat hepatocytes on insulin action and insulin receptor structure and function. Twenty hours of preincubation with glyburide (1 microgram/mL) resulted in increased insulin (1 X 10(-7) mol/L) stimulation of [14C] acetate incorporation into lipids and [14C] alpha-aminoisobutyric acid uptake without any change in basal activity. Ciglitazone (1 microgram/mL) was without any effect. Glyburide's actions were mediated without altering the following: (1) 125I-insulin binding; (2) the electrophoretic mobility of the affinity labeled alpha-subunit or the autophosphorylated beta-subunit of the insulin receptor; and (3) the insulin-stimulated insulin receptor kinase activity using histone or the beta-subunit of the insulin receptor as phosphoacceptors. These data suggest that the action of sulfonylureas is distal to the insulin receptor tyrosine kinase. Ciglitazone in vitro is ineffective in the liver, which suggests the peripheral tissues as the possible site of action.     

Opn5 is a UV-sensitive bistable pigment that couples with Gi subtype of G protein

Opn5 (neuropsin) belongs to an independent group separated from the other six groups in the phylogenetic tree of opsins, for which little information of absorption characteristics and molecular properties of the members is available. Here we show that the chicken Opn5 (cOpn5m) is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. The recombinant expression of cOpn5m in HEK 293s cells followed by the addition of 11-cis- and all-trans-retinal produced UV light-absorbing and visible light-absorbing forms, respectively. These forms were interconvertible by UV and visible light irradiations, respectively, indicating that cOpn5m is a bistable pigment. The absorption maxima of these forms were estimated to be 360 and 474 nm, respectively. The GTPγS binding assay clearly showed that the visible light-absorbing form having all-trans-retinal activates Gi type of G protein, whereas no Gt or Gq activation ability was observed. Immunohistochemical studies using an antibody against cOpn5m clearly showed that this pigment is localized within some types of amacrine cells and some cells in the ganglion cell layer of the retinas, the vast majority of cells in the pineal gland and serotonin-positive cells in the paraventricular organ. Because cOpn5m is the only UV-sensitive opsin among the opsins found so far in chicken, this study provides the molecular basis for UV reception in chicken.     

Increased insulin action in the rat after protein malnutrition early in life

Summary The effect of a limited period of low protein feeding in young rats on insulin secretion and insulin action during adult-age has been studied. Four-week-old rats were maintained for 4 weeks on isocaloric diets containing 5% protein (low protein) or 15% protein (control). The low protein rats gained weight at a considerably lower rate than the control rats. This was obtained in the absence of any decrease of spontaneous food intake. Basal plasma insulin levels were decreased (p<0.01) by 40% in low protein rats. However, the glucose-stimulated insulin secretion obtained in vivo after an i.v. glucose load remained normal. The basal plasma glucose level in the low protein rats was only marginally decreased (by 20%). The tolerance to i.v. glucose was found to be slightly enhanced in the low protein rats as compared to the control rats as shown by a significantly increased K value (p}<0.01). In vivo insulin action in the low protein rats was investigated using the euglycaemic-hyperinsulinaemic clamp technique in conjunction with isotopic measurements of glucose turnover. The overall glucose utilization rate was normal in the basal state but significantly increased (p<0.05) when measured at a submaximal plasma insulin level. The basal hepatic glucose production in the low protein rats was similar to that in the control rats. During the clamp studies, the suppression of endogenous glucose production was found to be similar in the low protein rats and the control rats but this was obtained at significantly lower (p<0.01) steady-state insulin levels in the low protein group than in the control group. In conclusion, the current results indicate that the modest improvement of glucose tolerance which is revealed in the low protein rats results from changes in the insulin action upon the target tissues: both the insulin-mediated glucose uptake by peripheral tissues and the ability of insulin to suppress hepatic glucose output are enhanced.     

Magnesium requirement for somatostatin inhibition of insulin secretion

Rat pancreas perfusions were performed using a perfusate with a fixed calcium concentration of 5 mEq/l and magnesium varying from 0 to 0.6 mEq/dl. Insulin secretion was stimulated by a constant glucose infusion of 300 mg/dl. This glucose concentration produces the typical biphasic insulin secretory response. We observed that in the absence of magnesium, somatostatin concentrations of 0.5 and 2.0 ng/ml were without effect on first phase insulin secretion. However, these same somatostatin levels produced 50% or more inhibition of insulin secretion in the presence of magnesium at 0.3 or 0.6 mEq/l. Similarly, in the absence of magnesium, somatostatin at 50 ng/ml failed to inhibit second phase insulin secretion, whereas this same somatostatin level produced about 50% inhibition of insulin secretion in the presence of magnesium at 0.3 mEq/l. Thus, altering perfusate magnesium concentrations without changing calcium is an important determinant of the degree of inhibition of secretion produced by somatostatin. In particular, in the absence of magnesium ion, somatostatin concentrations which would 'normally' produce 50% inhibition of secretion (ID50) are without effect. Therefore, magnesium ion is necessary for the full inhibitory effect of somatostatin to occur. These results suggest that inhibitors, as well as potentiators, of the insulin secretory process may act by altering intracellular/membrane calcium-magnesium ratios, but in opposite directions.     

In vivo insulin action in hepatocellular and cholestatic liver cirrhosis.

The in vivo dose response curve to insulin were studied, using an euglycemic insulin clamp technique, in 13 cirrhotic patients [8 with "hepatocellular" (HC) (nonalcoholics) and 5 with "cholestatic" (CHOL) cirrhosis] and 12 healthy controls (N). Subjects were studied in the basal state and during infusion of insulin at 3 different rates - 1, 3, 10 mU kg-1 min-1. Insulin responsiveness was similar in N and in HC, but it was 23% greater in CHOL (p less than 0.001). Insulin sensitivity was decreased in cirrhotics as compared with N but this difference was only significant (p less than 0.001) in HC. (ED50:62 + 5, 88 + 13 and 136 + 16 muu ml-1 in N, CHOL and HC respectively). Insulin clearance rate (ICR) was significantly (p less than 0.005) decreased in HC (1060 +/- 80, 996 +/- 95 and 776 +/- 128 ml sq m-1 ml-1 in N, CHOL and HC respectively. Basal hepatic glucose production (BHGP) was 39% lower in HC (p less than 0.005) and 24% lower in CHOL (p less than 0.05) than in N. Erythrocyte cholesterol phospholipid ratio was significantly elevated (p less than 0.001) in both groups of cirrhotic patients but was not correlated to specific metabolic changes described. In summary: i) intervariations in insulin dependent glucose metabolism were described in different cirrhotic groups; ii) basal hepatic glucose production and insulin clearance rate impaired in the different groups of cirrhotics; iii) the role of decreased cholesterol/phospholipid ratio on tissues glucose metabolism in cirrhotic patients should be further studied.