Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains

Na+ transport through the F0 domain of Na(+)-F1F0-ATPases involves the combined action of subunits c and a but the residues involved in Na+ liganding in subunit a are unknown. As a first step towards the identification of these residues, we have cloned and sequenced the gene encoding subunit a of the Na(+)-F1F0-ATPase of Acetobacterium woodii. This is the second sequence available now for this subunit from Na(+)-F1F0-ATPases. A comparison of subunit a from Na(+)-F1F0-ATPases with those from H(+)-translocating enzymes unraveled structural similarity in a C-terminal segment including the ultimate and penultimate transmembrane helix. Seven residues are conserved in this region and, therefore, likely to be involved in Na+ liganding.     

Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop

Protease-activated receptor 1 (PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (G(q/11), G(i/o), and G(12/13)) and their linked signaling pathways to regulate a broad range of pathophysiological processes. However, the molecular mechanisms whereby PAR1 interacts with multiple G proteins are not well understood. Whether PAR1 interacts with various G proteins at the same, different, or overlapping binding sites is not known. Here we investigated the functional and specific binding interactions between PAR1 and representative members of the G(q/11), G(i/o), and G(12/13) subfamilies. We report that G(q/11) physically and functionally interacts with specific amino acids within the second intracellular (i2) loop of PAR1. We identified five amino acids within the PAR1 i2 loop that, when mutated individually, each markedly reduced PAR1 activation of linked inositol phosphate formation in transfected COS-7 cells (functional PAR1-null cells). Among these mutations, only R205A completely abolished direct G(q/11) binding to PAR1 and also PAR1-directed inositol phosphate and calcium mobilization in COS-7 cells and PAR1-/- primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either G(o) or G(12), or their functional coupling to linked pertussis toxin-sensitive ERK phosphorylation and C3 toxin-sensitive Rho activation, respectively. In astrocytes, our findings suggest that PAR1-directed calcium signaling involves a newly appreciated G(q/11)-PLCε pathway. In summary, we have identified key molecular determinants for PAR1 interactions with G(q/11), and our findings support a model where G(q/11), G(i/o) or G(12/13) each bind to distinct sites within the cytoplasmic regions of PAR1.     

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.     

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.     

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.     

The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).     

Coxsackievirus and adenovirus receptor (CAR) is modified and shed in membrane vesicles

Vesicles shed by U87-MG cells contain coxsackievirus and adenovirus receptor (CAR) protein that has been posttranslationally modified. Relative to full-length CAR, migration of the vesicle-associated soluble CAR antigen (CARd6) on SDS-polyacrylamide gels indicated a loss of approximately 6 kDa. HeLa and END-HHV6 cells also shed a similar vesicle-associated CAR protein. Vesicles shed by U87-MG cells following stimulation with calcium and A23187 contained CARd6 similar to that present in vesicles shed constitutively. RD cells transfected to express full-length CAR produced CARd6, but cells that expressed CAR with a truncated cytoplasmic domain produced no equivalent to CARd6. In U87-MG cells, calpain activity was required for release of CARd6 with shed vesicles, and accumulation of CARd6 in cells that rounded up and released from the plastic substrate in response to A23187 treatment was blocked by N-ethylmaleimide. These experiments show that CAR, posttranslationally modified in the cytoplasmic domain, can be released with vesicles shed by cells. Posttranslational modification of the CAR cytoplasmic domain occurs during cell rounding and release from the culture substrate. This modified, vesicle-associated CAR was the principal form of soluble CAR released by the cells.     

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.     

Involvement of betagamma subunits of G(q/11) in muscarinic M(1) receptor potentiation of corticotropin-releasing hormone-stimulated adenylyl cyclase activity in rat frontal cortex

In the present study, we investigated the involvement of betagamma subunits of G(q/11) in the muscarinic M(1) receptor-induced potentiation of corticotropin-releasing hormone (CRH)-stimulated adenylyl cyclase activity in membranes of rat frontal cortex. Tissue exposure to either one of two betagamma scavengers, the QEHA fragment type II adenylyl cyclase and the GDP-bound form of the alpha subunit of transducin, inhibited the muscarinic M(1) facilitatory effect. Moreover, like acetylcholine (ACh), exogenously added betagamma subunits of transducin potentiated the CRH-stimulated adenylyl cyclase activity, and this effect was not additive with that elicited by ACh. Western blot analysis indicated the expression in frontal cortex of both type II and type IV adenylyl cyclases, two isoforms stimulated by betagamma subunits in synergism with activated G(s). The M(1) receptor-induced enhancement of the adenylyl cyclase response to CRH was counteracted by the G(q/11) antagonist GpAnt-2A but not by GpAnt-2, a preferential G(i/o) antagonist. In addition, the muscarinic facilitatory effect was inhibited by membrane preincubation with antiserum directed against the C terminus of the alpha subunit of G(q/11), whereas the same treatment with antiserum against either G(i1/2) or G(o) was without effect. These data indicate that in membranes of rat frontal cortex, activation of muscarinic M(1) receptors potentiates CRH-stimulated adenylyl cyclase activity through betagamma subunits of G(q/11).     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

G protein G alpha q/11 and G alpha i1,2 are activated by pancreastatin receptors in rat liver: studies with GTP-gamma 35S and azido-GTP-alpha-32P

In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S-GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively.     

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.     

A G(q/11)-coupled mutant histamine H(1) receptor F435A activated solely by synthetic ligands (RASSL)

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study Galpha(i) signaling in vivo (1, 2). Also, Galpha(s)-coupled G protein-coupled receptors have been engineered to generate Galpha(s)-coupled RASSLs (3, 4). In this study, we exploited the differences in binding pockets between different classes of H(1) receptor agonists and identified the first Galpha(q/11)-coupled RASSL. The mutant human H(1) receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H(1) receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H(1) receptors.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.     

Cross-linking of endothelin 1 and endothelin 3 to rat brain membranes: identification of the putative receptor(s)

Affinity-labeling experiments with 125I-endothelin derivatives using bifunctional cross-linking reagents were carried out in an attempt to identify the polypeptide component(s) of the endothelin/sarafotoxin receptors in rat brain tissues. In rat cerebellum, cortex, and caudate putamen, endothelin 1 specifically labeled a major component with a molecular mass of around 53,000. In the same tissues endothelin 3 specifically labeled, in addition to the 53,000 band, a band of molecular mass of 38,000. This result clearly indicates that in the brain the endothelin binding site resides within a polypeptide of apparent Mr = 53,000. The possible presence of receptor subtypes is discussed with reference also to the reported identification of endothelin receptors in chick cardiac membrane and in rat mesangial cells.     

Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G(q/11)-dependent transactivation of the epidermal growth factor receptor

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.     

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.     

Possible interaction of alpha 1-adrenergic receptor with pertussis-toxin-sensitive guanine-nucleotide-binding regulatory proteins (G proteins) responsible for phospholipase C activation in rat liver plasma membranes

Islet-activating protein (IAP; pertussis toxin) was employed to test the hypothesis that IAP-sensitive GTP-binding regulatory proteins (G proteins) are coupled with alpha 1-adrenergic receptor in rat liver plasma membranes. The high-affinity state of the binding of alpha 2-adrenergic agonist, which is known to be coupled with IAP-sensitive G protein, was abolished in IAP-treated plasma membranes. IAP treatment of plasma membranes could also diminish the high-affinity state of the alpha 1-adrenergic receptor for the agonist. Restoration of the high-affinity state of the alpha 1-adrenergic receptor for the agonist occurred on reconstitution of the bovine brain IAP-sensitive G proteins. The alpha 1-adrenergic receptor agonist stimulated inositol triphosphate (InsP3) production from [3H]inositol-labeled liver plasma membranes in a concentration-dependent manner. IAP treatment also decreased alpha 1-adrenergic-agonist-induced InsP3 production but not completely. From these results, we concluded that there is a possibility that both IAP-sensitive and IAP-insensitive G proteins were involved in alpha 1-adrenergic-receptor-stimulated phospholipase C activation in rat liver plasma membranes.