guanosine-diphosphate and aluminum-fluoride

Guanine nucleotide-binding (G) proteins, which cycle between a GDP- and a GTP-bound conformation, are conventionally regulated by GTPase-activating proteins (GAPs) and guanine nucleotide-exchange factors (GEFs), and function by interacting with effector proteins in the GTP-bound 'on' state. Here we present another class of G proteins that are regulated by homodimerization, which we would categorize as G proteins activated by nucleotide-dependent dimerization (GADs). This class includes proteins such as signal recognition particle (SRP), dynamin, septins and the newly discovered Roco protein Leu-rich repeat kinase 2 (LRRK2). We propose that the juxtaposition of the G domains of two monomers across the GTP-binding sites activates the biological function of these proteins and the GTPase reaction.

Topics: Aluminum Compounds; Animals; Binding Sites; Dimerization; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Molecular; Plant Proteins; Protein Structure, Quaternary

The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.

Topics: Aluminum Compounds; Bacterial Proteins; Base Sequence; Catalytic Domain; Escherichia coli; Escherichia coli Proteins; Fluorides; GTP Phosphohydrolase Activators; GTP Phosphohydrolases; Guanosine Diphosphate; Hydrolysis; Molecular Sequence Data; Receptors, Cytoplasmic and Nuclear; RNA; RNA, Bacterial; Sequence Analysis, DNA; Signal Recognition Particle

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.

Topics: Aluminum Compounds; Animals; Bacterial Proteins; Binding Sites; Catalysis; Cell Line; Escherichia coli; Eye Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Fluorides; Green Fluorescent Proteins; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; GTP-Binding Protein Regulators; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Inhibitory Concentration 50; Insecta; Kinetics; Luminescent Proteins; Mutation; Phosphoproteins; Protein Binding; RGS Proteins; Spectrometry, Fluorescence; Type C Phospholipases

We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.

Topics: Actins; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Aluminum Compounds; Animals; Biological Transport; Cell Line; Cricetinae; Cytoskeleton; Fluorides; GTPase-Activating Proteins; Guanosine Diphosphate; HeLa Cells; Humans; Mutation; Phospholipids; Protein Binding; Recombinant Proteins

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

Topics: Aluminum Compounds; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Dimerization; Fluorides; Fluorometry; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Models, Molecular; Molecular Conformation; Protein Binding; Receptors, Cytoplasmic and Nuclear; RNA, Bacterial; Signal Recognition Particle; Thermus

FtsZ, the prokaryotic homologue of tubulin, is an essential cell division protein. In the cell, it localizes at the center, forming a ring that constricts during division. In vitro, it binds and hydrolyzes GTP and polymerizes in a GTP-dependent manner. We have used atomic force microscopy to study the structure and dynamics of FtsZ polymer assembly on a mica surface under buffer solution. The polymers were highly dynamic and flexible, and they continuously rearranged over the surface. End-to-end joining of filaments and depolymerization from internal zones were observed, suggesting that fragmentation and reannealing may contribute significantly to the dynamics of FtsZ assembly. The shape evolution of the restructured polymers manifested a strong inherent tendency to curve. Polymers formed in the presence of non-hydrolyzable nucleotide analogues or in the presence of GDP and AlF(3) were structurally similar but showed a slower dynamic behavior. These results provide experimental evidence supporting the model of single-strand polymerization plus cyclization recently proposed to explain the hydrodynamic behavior of the polymers in solution.

Topics: Adsorption; Aluminum Compounds; Aluminum Silicates; Chemical Phenomena; Chemistry, Physical; Escherichia coli; Escherichia coli Proteins; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen-Ion Concentration; Microscopy, Atomic Force; Polymers; Solutions

The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-alpha (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT*), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of alphaT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT*(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP-bound forms of alphaT*, alphaT*(R238A) and alphaT*(R238K), the alphaT*(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on alphaT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of alphaT*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative G alpha subunit that can effectively block signaling by G protein-coupled receptors.

Topics: Aluminum Compounds; Animals; Arginine; Catalysis; Cattle; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Fluorides; Genes, Dominant; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Models, Biological; Models, Molecular; Mutation; Nucleotides; Point Mutation; Protein Conformation; Recombinant Fusion Proteins; Recombinant Proteins; Retina; Rhodopsin; Signal Transduction; Spectrometry, Fluorescence; Time Factors; Transducin; Trypsin

To determine the role of the myristoylated amino terminus of Galpha in G protein activation, nine individual cysteine mutations along the myristoylated amino terminus of Galpha(i) were expressed in a functionally Cys-less background. Thiol reactive EPR and fluorescent probes were attached to each site as local reporters of mobility and conformational changes upon activation of Galpha(i)GDP by AlF(4)(-), as well as binding to Gbetagamma. EPR and steady state fluorescence anisotropy are consistent with a high degree of immobility for labeled residues in solution all along the amino terminus of myristoylated Galpha(i). This is in contrast to the high mobility of this region in nonmyristoylated Galpha(i) [Medkova, M., et al. (2002) Biochemistry 41, 9962-9972]. Steady state fluorescence measurements revealed pronounced increases in fluorescence upon activation for residues 14-17 and 21 located midway through the 30-amino acid stretch comprising the amino-terminal region. Collectively, the data suggest that myristoylation is an important structural determinant of the amino terminus of Galpha(i) proteins.

Topics: Aluminum Compounds; Cysteine; DNA Primers; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Fluorides; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Models, Molecular; Mutagenesis, Site-Directed; Myristic Acid; Protein Binding; Protein Conformation; Spin Labels; Structure-Activity Relationship; Sulfhydryl Compounds

The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP.

Topics: Aluminum Compounds; Animals; Beryllium; Binding Sites; Brain Chemistry; Cattle; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Male; Microscopy, Video; Microtubules; Phosphates; Phosphorus Radioisotopes; Polymers; Sea Urchins; Spermatozoa; Thermodynamics; Tubulin

Human MxA protein is a member of the interferon-induced Mx protein family and an important component of the innate host defense against RNA viruses. The Mx family belongs to a superfamily of large GTPases that also includes the dynamins and the interferon-regulated guanylate-binding proteins. A common feature of these large GTPases is their ability to form high molecular weight oligomers. Here we determined the capacity of MxA to self-assemble into homo-oligomers in vitro. We show that recombinant MxA protein assembles into long filamentous structures with a diameter of about 20 nm at physiological salt concentration as demonstrated by sedimentation assays and electron microscopy. In the presence of guanosine nucleotides the filaments rearranged into rings and more compact helical arrays. Our data indicate that binding and hydrolysis of GTP induce conformational changes in MxA that may be essential for viral target recognition and antiviral activity.

Topics: Aluminum Compounds; Dimerization; Dynamins; Escherichia coli; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Microscopy, Electron; Myxovirus Resistance Proteins; Protein Binding; Protein Conformation; Proteins; Recombinant Proteins; Salts

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP gamma S, GDP beta S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP gamma S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP gamma S could be antagonised by pre-treatment of hyphae with GDP beta S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP gamma S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP gamma S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect.

Topics: Aluminum Compounds; Basidiomycota; Brefeldin A; Cytoskeleton; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Hyphae; Protein Synthesis Inhibitors; Thionucleotides; Transport Vesicles; Vacuoles

The formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg(2+) and F(-) but without Al(3+). The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF(4)(-). We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF(-) possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3(-) may be the reagent of choice for studying phosphoryl transfer reactions.

Topics: Aluminum Compounds; Fluorides; Guanosine Diphosphate; Magnesium Compounds; Models, Molecular; Phosphorus; rho GTP-Binding Proteins; Spectrometry, X-Ray Emission

Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.

Topics: Aluminum Compounds; Amino Acid Sequence; Bacterial Toxins; Binding Sites; Catalysis; Crystallography, X-Ray; Down-Regulation; Fluorides; GTPase-Activating Proteins; Guanosine Diphosphate; Histidine Kinase; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Folding; Protein Kinases; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa; rac GTP-Binding Proteins; Sequence Alignment; Structure-Activity Relationship

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl- conductance (CFTR G(Cl)) in the native sweat duct (SD). We permeabilized the basolateral membrane with alpha-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G(Cl) activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-gamma-S (100 microm) also activates CFTR G(Cl) in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-gamma-S increased CFTR G(Cl) by 44 +/- 20 mS/cm(2) (mean +/- se; n = 7). GDP (10 mm) inhibited G-protein activation of CFTR G(Cl) even in the presence of GTP-gamma-S. The heterotrimeric G-protein activator (AlF(4-) in the cytoplasmic bath activated CFTR G(Cl) (increased by 51.5 +/- 9.4 mS/cm(2) in the presence of 5 mm ATP without cAMP, n = 6), the magnitude of which was similar to that induced by GTP-gamma-S. Employing immunocytochemical-labeling techniques, we localized Galphas, Galphai, Galphaq, and Gbeta at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G(Cl) in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G(Cl) activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G(Cl) activity.

Topics: Adenosine Triphosphate; Adult; Aluminum Compounds; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Humans; Immunohistochemistry; In Vitro Techniques; Male; Mutation; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Sweat Glands

We have previously shown that not only G protein-coupled receptor kinase (GRK) 2, but also a catalytically inactive Lys220Trp GRK2 decreases endothelin (ET)-1-induced inositol 1,4,5-trisphosphate (IP3) formation, and demonstrated the presence of phosphorylation-independent desensitization mechanism. To clarify the role of GRK2 other than that as a kinase, we characterized an RGS (regulator of G protein signaling)-like domain in the amino-terminus of GRK2. Both GRK2(1-181) and GRK2(54-174) suppressed Ca2+ responses induced by angiotensin II (Ang II) and ET-1, and bound directly with Galphaq but not Galphas nor Galphai3 in the presence of GDP and AlF4-. These results demonstrate that GRK2 regulates Gq-mediated signaling negatively by direct interaction between its RGS domain and the transitional state of Galphaq, as well as through phosphorylation of activated receptors by its kinase domain.

Topics: Aluminum Compounds; Angiotensin II; beta-Adrenergic Receptor Kinases; Calcium; Cell Line; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Fluorides; Fura-2; GTP-Binding Protein Regulators; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Plasmids; Signal Transduction; Transfection

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

Topics: 3T3 Cells; Actins; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Aluminum Compounds; Amino Acid Sequence; Amino Acid Substitution; Animals; Arginine; Carrier Proteins; Cell Membrane; Cell Surface Extensions; Conserved Sequence; Cytoplasm; Cytoskeleton; Fluorides; GTPase-Activating Proteins; Guanosine Diphosphate; HeLa Cells; Humans; Mice; Molecular Sequence Data; Multigene Family; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Platelet-Derived Growth Factor; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity

Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.

Topics: Aluminum Compounds; Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Binding Sites; cdc42 GTP-Binding Protein; Crystallography, X-Ray; Dimerization; Evolution, Molecular; Fluorides; GTPase-Activating Proteins; Guanosine Diphosphate; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Tyrosine Phosphatases; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Salmonella typhimurium; Sequence Alignment; Signal Transduction

The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.

Topics: Alanine; Aluminum Compounds; Arginine; Catalysis; Conserved Sequence; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Humans; Lysine; Macromolecular Substances; ortho-Aminobenzoates; Protein Binding; Rho Factor

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.

Topics: Aluminum Compounds; Animals; Base Sequence; Cattle; Conserved Sequence; DNA Primers; Fluorides; Glutamic Acid; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Kinetics; Magnesium; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; Recombinant Proteins

hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.

Topics: Aluminum Compounds; Binding Sites; Binding, Competitive; Calorimetry; DNA-Binding Proteins; Fluorides; Fluorometry; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mutation; Recombinant Proteins; Thermodynamics

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

Topics: Aluminum Compounds; Animals; Binding Sites; COS Cells; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Models, Molecular; Mutation; Phenotype; Protein Conformation; Retinaldehyde; Rhodopsin; Rod Cell Outer Segment; Transducin

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

Topics: Aluminum Compounds; Amino Acid Sequence; Binding Sites; Catalysis; Cell Transformation, Neoplastic; Crystallography, X-Ray; Enzyme Activation; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Protein Structure, Secondary; Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction

Topics: Aluminum Compounds; Binding Sites; Catalysis; Crystallography, X-Ray; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Models, Molecular; Protein Conformation; Protein Structure, Secondary; Proteins; ras GTPase-Activating Proteins; ras Proteins; RGS Proteins

Internal administration of the G protein activator, guanosine-5'-o-(3-thiotriphosphate) (GTP gamma S) or aluminum fluoride (AIF) complex, produced an inward nonselective cation current (INS) at -55 mV. This current was rapidly diminished under conditions of high intracellular Mg2+ ([Mg2+] = 979 microM), the half decay time (T1/2) being 80 to 100 s. As [Mg2+] in AlF solutions decreased from 400 to 12 microM, the maximum amplitude of AlF-induced INS became larger and the current was diminished more slowly. The AlF INS in the presence of 12 microM Mg2+ reversed polarity at about +9 mV, irrespective of the extent of decline. Bath application of muscarine produced a sustained INS in the absence of AlF complex, but in its presence, the overall current comprising a spontaneously developed INS and muscarine-induced INS was rapidly diminished. Addition of vanadate (0.5 mM) to 979 microM Mg2+ -containing AlF solution mimicked the effects of low Mg2+ solution. Inversely, addition of alkaline phosphatase (40 units/ml) to 12 microM Mg2+ AlF solution reproduced the effects of high Mg2+ solution. It is suggested that AlF complex deactivates INS through facilitating an apparent activity of Mg2+ -dependent phosphatase.

Topics: Aluminum Compounds; Animals; Cations; Chromaffin System; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guinea Pigs; Ion Channels; Magnesium; Muscarine; Muscarinic Agonists; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Protein Phosphatase 2C

The effect of modifiers of guanine nucleotide-binding proteins (G proteins) on the frequency augmentation-potentiation of transmitter release were studied in the frog neuromuscular junction. Using Genetransfer as a carrier the mean quantal content of the endplate potential increased by penetration of GTP gamma S into the presynaptic nerve terminal. Neither GTP gamma S alone nor carrier alone had any effect. The relationship of log (mean quantal content) versus stimulation frequency changed from a single linear to a dual linear function, suggesting that the immediately releasable pool was modified. GDP beta S + carrier also had similar effects, but was less potent. Aluminium fluoride was without effect. Extracellularly recorded presynaptic nerve action potentials remained unchanged with GTP gamma S + carrier. Also, GTP gamma S + carrier did not affect the action potential nor the cytosolic Ca2+ concentration in differentiated NG108-15 hybrid cells. It is suggested that some smg-type G protein-dependent processes are involved in determining frequency augmentation-potentiation.

Topics: Action Potentials; Aluminum Compounds; Animals; Cell Line; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Neuromuscular Junction; Ranidae; Thionucleotides

We used the whole-cell patch-clamp technique to monitor changes in membrane capacitance (Cm) to study the influence of cytosolic concentration ([Cl-]i) on the secretory activity of rat melanotrophs. The sensitivity of the secretory machinery to Ca2+ was enhanced in the presence of a high [Cl-]i. The free concentration of Ca2+ required for half-maximal secretory activity was reduced from 3.2microM at 4mM [Cl-]i to 0.7microM at 154mM [Cl-]i. To study whether the modulation of secretory activity by Cl- involves guanosine 5'-triphosphate-(GTP-) binding proteins, cells were dialysed with non-hydrolysable GTP and GDP analogues, fluoroaluminate (AlF4(-)), or were pretreated with pertussis toxin. With guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S], 100microM) the maximal rate of Cm increase (dCm/dt) was enhanced at 4 and 14mM [Cl-]i, but it was not affected at 154mM [Cl-]i. In contrast, the secretory response, measured as a percentage of resting Cm 10min after the start of recordings, was reduced at 154mM [Cl-]i, but not affected at 4mM [Cl-]i. Only with 154mM [Cl-]i did intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) (GDP[beta-S], 500microM) inhibit dCm/dt as well as relative secretory responses. The presence of AlF4(-) (30microM) or a 7-h pretreatment of cells with pertussis toxin (250ng/ml) significantly reduced both the maximal dCm/dt and relative secretory responses, but only in the presence of 154mM [Cl-]i. Since the effects of GDP[beta-S], AlF4(-), and pertussis toxin pretreatment were only detected with a high [Cl-]i, we conclude that modulation by Cl- of secretory activity of rat melanotrophs is mediated through GTP-binding proteins. Furthermore, the effects of AlF4(-) and pertussis toxin indicate a role of heterotrimeric GTP-binding proteins in the secretory activity of melanotrophs.

Topics: Aluminum Compounds; Animals; Calcium; Cells, Cultured; Chlorides; Cytosol; Electrophysiology; Exocytosis; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Melanocytes; Patch-Clamp Techniques; Pertussis Toxin; Rats; Thionucleotides; Virulence Factors, Bordetella

We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan fluorescence, with the rate for the dissociation of holotransducin into its component subunits. Specifically, a beta gamma T complex that was labeled with 5-(iodoacetamido)fluorescein (IAF-beta gamma T) served as a donor for resonance energy transfer and an alpha T-GDP species labeled with eosin 5-isothiocyanate (EITC-alpha TGDP) served as the acceptor. The quenching of IAF-beta gamma T fluorescence emission by the addition of the EITC-alpha TGDP species, due to resonance energy transfer between the IAF and EITC moieities, ranged from 10% to 15%. The association of the transducin subunits was rapid (i.e., within the time period of mixing) and dose-dependent, yielding an apparent Kd of approximately 150 nM for the alpha TGDP/beta gamma T interaction. Unexpectedly, we find that the dissociation of IAF-beta gamma T from an aluminum fluoride-activated alpha TGDP/IAF-beta gamma T complex occurs prior to the onset of the intrinsic fluorescence changes in alpha T that accompany activation of this subunit. Thus, there are at least two structural changes in alpha T that result from the occupation of the gamma-phosphate position in the nucleotide binding cleft of alpha T by aluminum fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aluminum Compounds; Animals; Cattle; Energy Transfer; Eosine Yellowish-(YS); Fluoresceins; Fluorescent Dyes; Fluorides; Guanosine Diphosphate; Kinetics; Macromolecular Substances; Protein Conformation; Spectrometry, Fluorescence; Transducin

An ion-counterion interaction between the lysine of the NKXD motif in the GTPase domain and an aspartate in the inserted helical domain of alpha subunits of heterotrimeric G proteins, Lys-278 and Asp-158, respectively, of Gs alpha is shown to be essential for activation by AlF4- and partially so for interaction with beta gamma dimers and activation by GTP and receptor. However, this domain interaction is not required for activation by the non-hydrolyzable analog guanosine 5'-3-O-(thio)triphosphate. Proximity of the helical domain to the GTPase domain is thus involved in the fundamental inactive-->active transition of the protein in a way that further distinguishes alpha subunits of heterotrimeric G proteins from ras and ras-like GTPases that lack helical domains and are neither activated by AlF4- nor combine with beta gamma dimers.

Topics: Aluminum Compounds; Amino Acid Sequence; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Molecular Sequence Data

Aluminium fluoride (AIF-4) activates members of the heterotrimeric G-protein (G alpha beta gamma) family by binding to inactive G alpha.GDP near the site occupied by the gamma-phosphate in G alpha.GTP (ref. 3). Here we describe the crystal structure of transducin alpha.GDP activated with aluminium fluoride (Gt alpha.GDP.AIF-4.H2O) at 1.7 A, a resolution sufficient to establish the coordination geometry of the bound aluminium fluoride as well as the extensive network of direct and water-mediated interactions that stabilize it. These observations are derived from three independent representations in the asymmetric unit, eliminating any chance of drawing conclusions based on stereochemistry imposed by crystal packing. Surprisingly, aluminium fluoride activates G alpha.GDP by binding with a geometry resembling a pentavalent intermediate for GTP hydrolysis. The stabilizing interactions involve not only residues that interact with the gamma-phosphate in Gt alpha.GTP gamma S, but also conserved residues for GTPase activity. Thus the Gt alpha.GDP.AIF-4.H2O structure provides new insight into the mechanism of GTP hydrolysis.

Topics: Aluminum Compounds; Animals; Cattle; Computer Graphics; Crystallography, X-Ray; Fluorides; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Hydrolysis; Mutagenesis; Protein Conformation; Transducin; Water

To determine whether G proteins activate cardiac ATP-sensitive K+ (KATP) channels by regulating intracellular ATP (ATPi)-dependent gating, currents were measured in inside-out patches. When ATPi closed KATP channels, activators of endogenous G proteins, GTP (plus adenosine or acetylcholine), GTP gamma S, or AlF-4 stimulated channels, an effect prevented by GDP beta S. In the absence of ATPi, G protein activators were ineffective. Intracellular nucleoside diphosphates restored KATP channel openings after the "rundown" of spontaneous activity. Only when ATPi suppressed nucleoside diphosphate-induced openings, GTP gamma S or AlF-4 enhanced KATP channel activity. Active forms of exogenous G protein subunits (G alpha i-1, G alpha i-2, or G alpha o) activated only KATP channels closed by ATPi. G proteins stimulate cardiac KATP channels apparently by antagonizing ATPi-dependent inhibitory gating. Regulation of ligand-dependent gating represents a distinct type of G protein modulation of ion channels.

Topics: Acetylcholine; Adenosine; Adenosine Triphosphate; Aluminum Compounds; Animals; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guinea Pigs; Inosine Diphosphate; Ion Channel Gating; Potassium Channels; Thionucleotides; Uridine Diphosphate

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.

Topics: 3T3 Cells; Adenine Nucleotides; Aluminum Compounds; Animals; Arachidonic Acid; Avian Sarcoma Viruses; Benzoquinones; Cell Line, Transformed; Cholera Toxin; Enzyme Induction; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Kinetics; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Myristic Acid; Myristic Acids; Oncogene Protein pp60(v-src); Phosphatidylcholines; Phospholipase D; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Thionucleotides; Virulence Factors, Bordetella

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aluminum Compounds; Amino Acid Sequence; Animals; Baculoviridae; DNA Mutational Analysis; Enzyme Activation; Fluorescence; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Models, Molecular; Molecular Sequence Data; Moths; Protein Conformation; Recombinant Proteins; Rod Cell Outer Segment; Sequence Homology, Amino Acid; Signal Transduction; Transducin; Tryptophan

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

Topics: Aluminum; Aluminum Compounds; Amino Acid Sequence; Coated Pits, Cell-Membrane; Endocytosis; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Peptides; Receptors, Transferrin; Thionucleotides; Tumor Cells, Cultured; Wasp Venoms

The thermodynamics of tubulin assembly into Zn sheets have been studied, with special emphasis on the role of bound nucleotide and of GTP hydrolysis in polymerization. In contrast to microtubules, Zn sheets could be assembled from GDP-tubulin as well as from GTP-tubulin. Accordingly, no appreciable destabilization of the Zn sheets was observed following GTP hydrolysis and P(i) release, indicating that the binding of Zn2+ to tubulin has abolished the regulatory switch role played by GTP hydrolysis in tubulin assembly. As a consequence, the critical concentration for assembly of Zn sheets did not increase with tubulin concentration, a feature characteristic of microtubule assembly. Zn sheets do not bind P(i) analogs, indicating that the gamma-phosphate binding locus of the E-site of tubulin is occluded following GTP hydrolysis in these GDP-tubulin polymers. Nonlinear van't Hoff plots were obtained for assembly of Zn sheets in the presence of either GTP or GDP, consistent with a change in heat capacity. Enthalpy, entropy, and heat capacity changes had values similar to those reported for assembly of microtubules or polymerization of tubulin-colchicine, indicating that hydrophobic tubulin-tubulin interactions are of comparable size in these different polymers.

Topics: Aluminum; Aluminum Compounds; Animals; Beryllium; Brain; Colchicine; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Microscopy, Electron; Polymers; Rats; Subtilisins; Swine; Temperature; Thermodynamics; Tubulin; Zinc

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C yields the second messengers inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol. This activity is regulated by a variety of hormones through G protein pathways. However, the specific G protein or proteins involved has not been identified. The alpha subunit of a newly discovered pertussis toxin-insensitive G protein (Gq) has recently been isolated and is now shown to stimulate the activity of polyphosphoinositide-specific phospholipase C (PI-PLC) from bovine brain. Both the maximal activity and the affinity of PI-PLC for calcium ion were affected. These results identify Gq as a G protein that regulates PI-PLC.

Topics: Aluminum; Aluminum Compounds; Animals; Calcium; Cattle; Enzyme Activation; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; In Vitro Techniques; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Substrate Specificity

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.

Topics: Adenosine Diphosphate Ribose; Aluminum; Aluminum Compounds; Animals; Calcium; Cells, Cultured; Dinoprostone; Epidermal Growth Factor; ErbB Receptors; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Kidney Medulla; Kidney Tubules; Kidney Tubules, Collecting; Kinetics; Pertussis Toxin; Phosphatidylinositols; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Tetradecanoylphorbol Acetate; Thionucleotides; Virulence Factors, Bordetella

Activation of transducin-GDP by NaF is mainly mediated by aluminofluorde or beryllofluoride complexes acting as GTP gamma-phosphate analogs. In millimolar magnesium, NaF at concentrations above 3 mM is active even in the absence of aluminium or beryllium. This activation has a Hill coefficient of 3 with respect to F-, and its rate is linear with respect to Mg2+ concentrations above 2 mM. Upon fluoride dilution, inactivation rate is hundreds of times faster than for aluminofluoride-activated T alpha GDP. We propose that at high NaF concentrations, 3 hydrogen-bonded fluorides in the gamma-phosphate site of T alpha GDP entrap a magnesium counterion and this induces the transconformation to the T alpha GTP form.

Topics: Aluminum; Aluminum Compounds; Animals; Beryllium; Cattle; Enzyme Activation; Fluorides; Guanosine Diphosphate; Kinetics; Magnesium; Rod Cell Outer Segment; Spectrometry, Fluorescence; Transducin

Aluminum fluoride and beryllium fluoride complexes have previously been shown to bind tightly to F-ADP-actin and GDP-microtubules in competition with Pi and to mimic the XDP-Pi transient state of the polymerization. The structure of the bound complexes is investigated here in further detail. Using a fluoride ion-specific electrode, the number of fluoride atoms per aluminum or beryllium atom in the bound complex could be determined. The results indicate that AIF-4 and either BeF2(OH)-.H2O or BeF3-.H2O are the tightly bound species in both F-actin and microtubules. The dependences of the binding on pF and pH are consistent with this conclusion. The possible geometries of aluminum and beryllium fluorides in the gamma-phosphate subsite of the nucleotide are discussed in correlation with the catalytic mechanism of nucleotide hydrolysis.

Topics: Actins; Adenosine Diphosphate; Aluminum; Aluminum Compounds; Animals; Beryllium; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Microtubules; Muscles; Rabbits

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Aluminum; Aluminum Compounds; Animals; Binding Sites; Cell Membrane; DNA; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Lymphoma; Magnesium; Mice; Mutation; Protein Conformation; Trypsin; Tumor Cells, Cultured

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aluminum; Aluminum Compounds; Animals; Beryllium; Cattle; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Photoreceptor Cells; Protein Binding; Rod Cell Outer Segment

Fluoride activation of the cGMP cascade of vision requires the presence of aluminum, and is shown to be mediated by the binding of one A1F-4 to the GDP/GTP-binding subunit of transducin. The presence of GDP in the site is required: A1F-4 is ineffective when the site is empty or when GDP beta S is substituted for GDP. This sensitivity to the sulfur of GDP beta S suggests that A1F-4 is in contact with the GDP. Striking structural similarities between A1F-4 and PO3-4 lead us to propose that A1F-4 mimics the role of the gamma-phosphate of GTP.

Topics: Aluminum; Aluminum Compounds; Animals; Binding Sites; Cattle; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Light; Membrane Proteins; Phosphates; Photoreceptor Cells; Rod Cell Outer Segment; Sodium Fluoride; Thionucleotides; Transducin