Membrane GTPase Research Articles

Legionella pneumophila proteins that regulate Rab1 membrane cycling

Rab1 is a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells. The intracellular pathogen Legionella pneumophila subverts Rab1 function to create a vacuole that supports bacterial replication by a mechanism that is not well understood. Here we describe L. pneumophila proteins that control Rab1 activity directly. We show that a region in the DrrA (defect in Rab1 recruitment A) protein required for recruitment of Rab1 to membranes functions as a guanine nucleotide dissociation inhibitor displacement factor. A second region of the DrrA protein stimulated Rab1 activation by functioning as a guanine nucleotide exchange factor. The LepB protein was found to inactivate Rab1 by stimulating GTP hydrolysis, indicating that LepB has GTPase-activating protein activity that regulates removal of Rab proteins from membranes. Thus, L. pneumophila encodes proteins that regulate three distinct biochemical reactions critical for Rab GTPase membrane cycling to redirect Rab1 to the pathogen-occupied vacuole and to control Rab1 function.

Functional dissection of Rab GTPases involved in primary cilium formation

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

Homocysteine-induced biochemical stress predisposes to cytoskeletal remodeling in stretched endothelial cells

Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.

Paradigm lost: milton connects kinesin heavy chain to miro on mitochondria

The kinesin motor typically binds to cargo through its light chains. In this issue Glater et al. (p. 545) demonstrate a new type of linkage through the adapter protein, milton, and the mitochondrial membrane GTPase, miro. This is an important result because it represents a new mechanism of cargo binding and because miro's ability to bind GTP and calcium suggests that it is involved in the regulation of mitochondrial transport.

Lindane stimulates neutrophils by selectively activating phospholipase C and phosphoinositide-kinase activity

The organochlorine insecticide lindane is a known activator of neutrophil responses. In this work we delineated the biochemical pathways by which lindane stimulates neutrophil oxidant production. Plasma membrane GTPase activity was not stimulated by lindane, ruling out a role for lindane-induced activation of G-proteins or G-protein coupled receptors, whereas inhibition of phospholipase C inhibited lindane-induced oxidant production. Together these data pointed to phospholipase C as the direct target of lindane activation. Type I phosphoinositide 3-kinase was not significantly activated by lindane and an inhibitor of phosphoinositide 3-kinases inhibited oxidant production by only 40%. Thus, Type I phosphoinositide 3-kinase played a minor role, if any, in lindane-induced oxidant production. Lindane stimulated an increase in phosphatidylinositol phosphate suggesting a Type II or III phosphotidylinositol 3-kinase or phosphatidylinositol 4-kinase activity was also stimulated.

Cellular Localization, Oligomerization, and Membrane Association of the Hereditary Spastic Paraplegia 3A (SPG3A) Protein Atlastin

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.

Novel approaches to enhance the detection of receptor constitutive activity and inverse agonists

Many wild-type G-protein-coupled receptors display relatively low levels of constitutive activity and thus identification of ligands with inverse agonist activity can be difficult. Two assays are available that monitor receptor interaction with a G protein, GTPase activity assays and [ 35S]GTPγS binding assays. GTPase assays are, however, frequently limited by high background. The fraction of membrane GTPase activity contributed by the constitutive activity of a receptor can be enhanced by addition of a recombinant regulator of G protein signalling protein to the assay. Detection of inverse agonists is then improved markedly. [ 35S]GTPγS binding assays for the detection of inverse agonists have historically been limited to receptors that activate pertussis toxin-sensitive G proteins. Introduction of a selective immunoprecipitation step using a G protein subtype specific antiserum allows this approach to be used for all G protein families. Application of these novel approaches, combined with the use of receptor–G protein fusion proteins to optimise receptor to G protein information transfer allows quantitative analysis of the extent of enhancement of constitutive activity produced by mutation of receptor or G protein.

Amyloid precursor protein carboxy-terminal fragments modulate G-proteins and adenylate cyclase activity in Alzheimer’s disease brain

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer’s disease (AD) and age-matched control brains was compared. 10 μM APP(639–648)–APP(657–676) (PEP1) causes a fivefold stimulation in the [ 35S]GTPγS-binding to control hippocampal G-proteins. APP(657–676) (PEP2) and APP(639–648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649–669) (PEP3) showed no regulatory activity in the [ 35S]GTPγS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [ 35S]GTPγS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

Withdrawal of statin treatment abrogates stroke protection in mice.

Statins (3-hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase inhibitors) reduce stroke damage independent of lipid lowering by upregulation of endothelial nitric oxide synthase (eNOS). Acute withdrawal of statin treatment may suppress endothelial NO production and impair vascular function. To test this hypothesis, we treated 129/SV mice with atorvastatin (10 mg/kg) for 14 days and then withdrew treatment. Treatment with atorvastatin conferred stroke protection by 40% after filamentous occlusion of the middle cerebral artery followed by reperfusion. Withdrawal of statin treatment, however, resulted in the loss of stroke protection after 2 and 4 days. In mouse aortas and brain vasculature, statins upregulated eNOS message 2.3- and 1.7-fold, respectively, as measured by reverse transcription-polymerase chain reaction. Withdrawal of statins resulted in 5- and 2.7-fold downregulation of eNOS in aorta and brain, respectively, after 2 days. Statin treatment decreased RhoA GTPase membrane expression to 48%, while withdrawal of statins resulted in 4-fold increase of RhoA in the membrane. Moreover, platelet factor 4 and beta-thromboglobulin in plasma were significantly downregulated by statin treatment, but withdrawal of statins resulted in a 2.9- and 3.1-fold upregulation after 2 days, respectively. Thrombus formation induced by ligature of the inferior vena cava was significantly reduced by statin treatment. When statin treatment was withdrawn, however, protection was lost between 2 and 4 days. Acute termination of statin treatment results in a rapid loss of protection in mouse models of cerebral ischemia and thrombus formation independent of lipid lowering. In patients with acute or impending stroke, withdrawal of statins may impair outcome.

Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ("desensitized") by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various beta-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of beta-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the beta-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from beta-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that beta-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.

Enhanced detection of receptor constitutive activity in the presence of regulators of G protein signaling: applications to the detection and analysis of inverse agonists and low-efficacy partial agonists.

Fusion proteins between the human 5-hydroxytryptamine (5-HT)(1A) receptor and either wild type or certain pertussis toxin-resistant forms of G(o1)alpha and G(i1)alpha display constitutive GTPase activity that can be inhibited by the inverse agonist spiperone. Addition of recombinant regulator of G protein signaling (RGS) 1 or RGS16 to membranes expressing these fusion proteins resulted in elevation of this constitutive GTPase activity without significantly altering the binding affinity of antagonist/inverse agonist ligands. For a 5-HT(1A) receptor-(Cys(351)Ile)G(o1)alpha fusion protein the increase in basal GTPase activity was greater than 4-fold. Enzyme kinetic analysis demonstrated that the effect of RGS1 was as a GTPase-activating protein for the fusion construct. In the presence of the RGS proteins, both agonists and inverse agonists produced much more robust regulation of high-affinity GTPase activity than in their absence. This allowed detection of the partial agonist nature of WAY100635, which has been described previously as a neutral antagonist at the 5-HT(1A) receptor. Of a range of ligands studied, only haloperidol functioned as a neutral ligand in the presence of RGS1. These studies show that addition of a recombinant RGS protein provides a simple and novel means to elevate the fraction of basal membrane GTPase activity contributed by the constitutive activity of a receptor. By so doing, it also greatly enhances the ability to detect and analyze the effects of inverse agonists and to discriminate between neutral ligands and those with low levels of positive intrinsic efficacy.

Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha (1E, 3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7- oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.

Modulation of phospholipase C pathway in rat cerebral cortex during aging

The regulation of the α 1-adrenoceptor-G protein-phospholipase C (PLC) cascade was investigated in rat cerebral cortex at adult (6-month-old) and senescent (24-month-old). Norepinephrine (NE)-stimulated inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3] production was enhanced 30% during aging. Moreover, maximal NE (50 μM) stimulation was much more effective in stimulating G protein low-K m GTPase in cortical membranes from old than adult rats. Immunoreactive G protein subunits (Gqα, Giα, Goα and Gcommonβ) and PLC-β 1 isozyme were detected in all membrane preparations. No changes in the G protein subunits and PLC-β 1 expression were observed with aging. Nanomolar concentration of Gpp[NH]p inhibited basal Ins(1,4,5)P 3 production with a maximum inhibition of 25% in both adult and aged cortical membranes. In contrast, 100 μM Gpp[NH]p-induced stimulation of Ins(1,4,5)P 3 production was potentiated with aging. The two principal divergent pathways of old cortical Ins(1,4,5)P 3 production resulting in the activation and inhibition of PLC-β 1 activity are abolished by treatment of the membranes with 1 μM U-73122, a putative PLC-β inhibitor. These results suggest that the cortical PLC-β 1 isozyme activity may be regulated by both inhibitory and stimulatory G proteins-mediated mechanisms, and that the altered PLC-β 1 dual regulatory systems could be involved in the pathogenesis of brain aging.

Identification of loci essential for the growth of Helicobacter pylori under acidic conditions.

To persist in the hostile acidic environment of the stomach, Helicobacter pylori must survive acid shock and grow at acidic pH. Of a library of 1250 random mutants screened for isolates unable to grow at low pH, 10 mutants were detected that were unable to grow at pH 4.8. However, all 10 mutants were resistant to acid shock. Four mutants had an insertion in genes of unknown function. One mutant was affected in lepA, an orthologue of a membrane GTPase. Three mutants were disrupted in loci involved in the transport of H(+) ions or other cations (FRaseI, czcA, and aldo-keto reductase). Two mutants were affected in loci that contribute to acid resistance in other microorganisms (uvrA and atpF'). Thus, at least 10 loci not related to urease are essential for the growth of H. pylori under acidic conditions and should be critical for lifelong infection by this pathogen.

The major protein import receptor of plastids is essential for chloroplast biogenesis.

Light triggers the developmental programme in plants that leads to the production of photosynthetically active chloroplasts from non-photosynthetic proplastids. During this chloroplast biogenesis, the photosynthetic apparatus is rapidly assembled, mostly from nuclear-encoded imported proteins, which are synthesized in the cytosol as precursors with cleavable amino-terminal targeting sequences called transit sequences. Protein translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes recognize these transit sequences, leading to the precursors being imported. The Toc complex in the pea consists of three major components, Toc75, Toc34 and Toc159 (formerly termed Toc86). Toc159, which is an integral membrane GTPase, functions as a transit-sequence receptor. Here we show that Arabidopsis thaliana Toc159 (atToc159) is essential for the biogenesis of chloroplasts. In an Arabidopsis mutant (ppi2) that lacks atToc159, photosynthetic proteins that are normally abundant are transcriptionally repressed, and are found in much smaller amounts in the plastids, although ppi2 does not affect either the expression or the import of less abundant non-photosynthetic plastid proteins. These findings indicate that atToc159 is required for the quantitative import of photosynthetic proteins. Two proteins that are related to atToc159 (atToc120 and atToc132) probably help to maintain basal protein import in ppi2, and so constitute components of alternative, atToc159-independent import pathways.

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic alpha-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase.

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Galphaq-phospholipase C-inositol 1, 4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A2 (TXA2). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA2 receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA2-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor-G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA2 receptors in vitro, but not Galphaq copurifying with the TXA2 receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA2 receptors by cyclic GMP was demonstrated from 32P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA2 receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA2 receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both alpha and beta forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA2 receptor. These data identify TXA2 receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.

Opioid Inhibition of Adenylyl Cyclase in Membranes from Pertussis Toxin-Treated NG108-15 Cells

Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of δ opioid receptor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential mechanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-32P]NAD+-labeling of G proteins and agonist stimulation of low Km GTPase in membranes. Although PTX-treatment decreased the maximal opioid inhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of adenylyl cyclase in untreated membranes was GTP-dependent), but was eliminated by hydrolysis-resistant guanine nucleotide analogs, indicating that G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [35S]GTPγS binding in the presence of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results suggest that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound opioid receptors without involvement of guanine nucleotide exchange.

CD14-dependent mechanism for endotoxin-mediated nitric oxide synthesis in murine macrophages.

Endotoxin-mediated macrophage synthesis of nitric oxide (NO) is associated with immune effector function, intercellular communication, leukocyte adhesion, vascular integrity, and neurotransmission. However, little is known of the cellular receptor and signal transduction pathway by which endotoxin induces NO production. With the use of a model of ANA-1 murine macrophages, we stimulated NO production by incubation with increasing concentrations of endotoxin and 5% fetal calf serum. In selected instances, the anti-CD14 antibody, ED9, was added. Endotoxin-mediated NO synthesis was dependent on CD14 function and the presence of an additional serum factor. Endotoxin treatment increased plasma membrane GTPase activity and 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding. Conversely, coincubation of cells with endotoxin and the heterotrimeric G protein inhibitors, suramin and guanosine 5'-O-(2-thiodiphosphate) trilithium salt, was associated with decreased NO synthesis, plasma membrane GTPase activity, and [35S]GTP gamma S binding. Blockade of CD14 or G protein function was associated with ablation of endotoxin-mediated inducible NO synthase (iNOS) protein expression, iNOS mRNA levels, and iNOS gene transcription, as determined by immunoblot, reverse transcriptase-polymerase chain reaction, and nuclear run-on analyses, respectively. These results indicate that endotoxin-mediated NO synthesis is a CD14-heterotrimeric G protein-dependent process.

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha-subunit, GtalphaGDPAlF4-, while its binding to GtalphaGTPgammaS or GtalphaGDP was weak. hRGSr and Pgamma did not compete for the interaction with GtalphaGDPAlF4-. Affinity of the Pgamma-GtalphaGDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtalphaGDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PgammaBC). Binding of hRGSr to GtalphaGDPAlF4- complexed with PgammaBC resulted in a maximal approximately 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtalphaGDPAlF4- (Kd 35 nM). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1. Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates exceeded 0.7 s-1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.