Basal GTPase Activity Research Articles

Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin.

Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Commonly, phosphorylation of S579 is considered activating, while S600 phosphorylation is considered inhibiting. However, direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. Here, we compare effects of S579 and S600 phosphorylation on purified Drp1, using phosphomimetic mutants and in vitro phosphorylation. Both phosphomimetic mutants are shifted toward smaller oligomers. Both phosphomimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wildtype Drp1, both S579D and S600D phosphomimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant (K38A) lacking GTPase activity stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.

A highly-sensitive high throughput assay for dynamin's basal GTPase activity.

Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin’s GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin’s stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin’s basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several “hits” that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin’s basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for more potent and chemically desirable dynamin inhibitors.

Cytoplasmic interaction of the tumour suppressor protein hSNF5 with dynamin-2 controls endocytosis

Human SNF5 (hSNF5; INI1, SMARCB1 or BAF47) is a component of the human SWI/SNF chromatin remodelling complex and a tumour suppressor mutated in rhabdoid tumours. It also associates with the integrase of the human immunodeficiency virus (HIV)-1. We show by fluorescence loss in photobleaching that hSNF5 is constantly shuttling between the nucleus and the cytoplasm, raising the question of what the role of hSNF5 is in the cytoplasm. Here, we demonstrate that hSNF5 directly interacts with the GTPase dynamin-2 (DNM2) in the cytoplasm. DNM2 is a large GTPase involved in endocytosis and vesicle dynamics, which has been related to HIV-1 internalization. We show that hSNF5 colocalizes with DNM2 in endocytic vesicles. Depletion of hSNF5, but not of other components of the SWI/SNF complex, destabilizes DNM2 and impairs DNM2-dependent endocytosis. Furthermore, we show that hSNF5 inhibits assembly-stimulated DNM2 GTPase activity but not basal GTPase activity in vitro. Altogether, these results indicate that hSNF5 affects both the stability and the activity of DNM2, uncovering an unexpected role of hSNF5 in modulating endocytosis, and open new perspectives in understanding the role of hSNF5 in tumour genesis.

Dynamin-Catalyzed Membrane Fission Requires Coordinated GTP Hydrolysis

Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.

Structures of the Human GTPase MMAA and Vitamin B12-dependent Methylmalonyl-CoA Mutase and Insight into Their Complex Formation

Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.

SNX18 shares a redundant role with SNX9 and modulates endocytic trafficking at the plasma membrane

SNX18 and SNX9 are members of a subfamily of SNX (sorting nexin) proteins with the same domain structure. Although a recent report showed that SNX18 and SNX9 localize differently in cells and appear to function in different trafficking pathways, concrete evidence regarding whether they act together or separately in intracellular trafficking is still lacking. Here, we show that SNX18 has a similar role to SNX9 in endocytic trafficking at the plasma membrane, rather than having a distinct role. SNX18 and SNX9 are expressed together in most cell lines, but to a different extent. Like SNX9, SNX18 interacts with dynamin and stimulates the basal GTPase activity of dynamin. It also interacts with neuronal Wiskott-Aldrich syndrome protein (N-WASP) and synaptojanin, as does SNX9. SNX18 and SNX9 can form a heterodimer and colocalize in tubular membrane structures. Depletion of SNX18 by small hairpin RNA inhibited transferrin uptake. SNX18 successfully compensates for SNX9 deficiency during clathrin-mediated endocytosis and vice versa. Total internal reflection fluorescence microscopy in living cells shows that a transient burst of SNX18 recruitment to clathrin-coated pits coincides spatiotemporally with a burst of dynamin and SNX9. Taken together, our results suggest that SNX18 functions with SNX9 in multiple pathways of endocytosis at the plasma membrane and that they are functionally redundant.

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin's GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin's basal GTPase activity and utilized it to define the structural framework that mediates GED's association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.

The Dynamin-Related Protein Mgm1p Assembles into Oligomers and Hydrolyzes GTP To Function in Mitochondrial Membrane Fusion

Mitochondrial dynamics resulting from competing membrane fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1p, a dynamin-related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous studies suggest that Mgm1p mediates mitochondrial inner membrane fusion in a manner similar to that of other dynamin proteins that use GTP hydrolysis and oligomerization to induce structural changes in lipid bilayers; however, a direct demonstration of these activities has yet to be presented. Here we show that purified Mgm1p forms low-order oligomers that are dependent on protein concentration, suggesting a dynamic and reversible interaction. We further demonstrate that Mgm1p has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in conserved motifs of the GTPase domain show markedly reduced or diminished GTPase activity. A mutation in the GTPase effector domain, involved in assembly and assembly-stimulated GTP hydrolysis, has basal GTPase activity similar to that of wild-type Mgm1p but has a weaker propensity to form oligomers. Finally, our data indicate that Mgm1p interacts specifically with negatively charged phospholipids found in mitochondrial membranes, and point mutations in the predicted lipid-binding domain abrogate these interactions. These findings suggest the presence of a putative lipid-binding domain, providing insight into how this protein mediates inner membrane fusion. Together, these data indicate that Mgm1p mediates fusion through oligomerization, GTP hydrolysis, and lipid binding in a manner similar to those of other dynamin mechanoenzymes.

Functional reconstitution of the human chemokine receptor CXCR4 with Gi/Go-proteins in Sf9 insect cells

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the G(i)/G(o)-family. CXCR4 plays a role in the pathogenesis of autoimmune diseases, human immunodeficiency virus infection and various tumors, fetal development as well as endothelial progenitor and T-cell recruitment. To this end, most CXCR4 studies have focused on the cellular level. The aim of this study was to establish a reconstitution system for the human CXCR4 that allows for the analysis of receptor/G-protein coupling at the membrane level. We wished to study specifically constitutive CXCR4 activity and the G-protein-specificity of CXCR4. We co-expressed N- and C-terminally epitope-tagged human CXCR4 with various G(i)/G(o)-proteins and regulator of G-protein signaling (RGS)-proteins in Sf9 insect cells. Expression of CXCR4, G-proteins, and RGS-proteins was verified by immunoblotting. CXCR4 coupled more effectively to Galpha(i1) and Galpha(i2) than to Galpha(i3) and Galpha(o) and insect cell G-proteins as assessed by SDF-1alpha-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1alpha-stimulated GTP hydrolysis. SDF-1alpha stimulated [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) binding to Galpha(i2). RGS4 did not enhance GTPgammaS binding. Na(+) salts of halides did not reduce basal GTPase activity. The bicyclam, 1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100), acted as CXCR4 antagonist but was devoid of inverse agonistic activity. Halides reduced the maximum SDF-1alpha-stimulated GTP hydrolysis in the order of efficacy I(-) > Br(-) > Cl(-). In addition, salts reduced the potency of SDF-1alpha at activating GTP hydrolysis. From our data, we conclude the following: (1) Sf9 cells are a suitable system for expression of functionally intact human CXCR4; (2) Human CXCR4 couples effectively to Galpha(i1) and Galpha(i2); (3) There is no evidence for constitutive activity of CXCR4; (4) RGS-proteins enhance agonist-stimulated GTP hydrolysis, showing that GTP hydrolysis becomes rate-limiting in the presence of SDF-1alpha; (5) By analogy to previous observations made for the beta(2)-adrenoceptor coupled to G(s), the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of G(i)-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.

Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis

The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.

Pressure effects on the GTPase activity of brain membrane G proteins of deep-living marine fishes

In marine fishes, heterotrimeric guanyl nucleotide binding proteins (G proteins), which couple cell surface membrane receptors to their effector elements, are sensitive to hydrostatic pressure. The intrinsic high affinity GTPase activity of the α subunits of G proteins in three signaling systems coupled to adenylyl cyclase, the A 1 adenosine receptor, the muscarinic cholinergic receptor and the β-adrenergic receptor, was tested at pressures up to 340 atm. Brain membrane preparations from four members of the deep-sea teleost fish family Macrouridae were studied. Coryphaenoides armatus, C. filifer, C. rupestris and Macrourus berglax have depth distributions which together span 100–5810 m. Increased pressure inhibited basal GTPase activity only in M. berglax, which of the four species has the shallowest center of abundance. Increased hydrostatic pressure did not alter the response of GTPase activity to the β-adrenergic receptor agonist isoproterenol. Increased pressure decreased the stimulation of GTPase activity by the A 1 adenosine receptor agonist cyclopentyladenosine (CPA) in C. armatus and M. berglax, and by the muscarinic cholinergic receptor agonist carbamyl choline in C. armatus, C. filifer and M. berglax. Decreased agonist-stimulation of the GTPase activity at elevated pressure may result from pressure-induced changes in conformational states or inhibition of agonist binding. The binding of the non-hydrolyzable GTP analog guanosine 5′-[γ-thio]triphosphate (GTP[S]) in response to CPA was determined at 5 °C and atmospheric pressure. Six macrourid species and a morid were studied. The halftime ( t 1/2) values for GTP[S] binding, ranging from 20.8 to 40.9 min, are similar to values previously reported for two other cold-adapted fishes.

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.

The Regulator of G Protein Signaling RGS4 Selectively Enhances α2A-Adreoreceptor Stimulation of the GTPase Activity of Go1α and Gi2α

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.

Thrombin Stimulates Pertussis Toxin-Sensitive and -Insensitive GTPase Activitiesand ADP-Ribosylation of Gi in Human Neuroblastoma SH-EP

Kinetic interaction between thrombin receptor and G proteins was investigated in human epithelial neuroblastoma cell line, SH-EP. In these cells, both α-thrombin and SFLLRNP (one-letter amino-acid code) stimulated GTPase activity and enhanced cholera toxin-catalyzed ADP-ribosylation of G_i2 in a concentration-dependent manner. Basal GTPase activity was attenuated by pertussis toxin treatment by 35%, however, agonist stimulation was preserved significantly. These results together indicated that thrombin receptor simultaneously activates G_i2 and PTX-insensitive G protein(s).

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery.

Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition

Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [gamma-32P]GTP and subjected to physiological levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0-30 dynes/cm2) activated G proteins in dose-dependent manner (0.48-4.6 pmol/min per microg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.

Abscisic acid induction of GTP hydrolysis in maize coleoptile plasma membranes

Since receptor-coupled G proteins increase GTP hydrolysis (GTPase) activity upon ligands binding to the receptor, a study was undertaken to determine if abscisic acid (ABA) induced such an effect. Plasma membranes isolated from etiolated maize (Zea mays L.) coleoptiles were enriched in GTPase activity relative to microsomal fractions. Vanadate was included in the assay to inhibit the high levels of vanadate sensitive low affinity GTPases present. Under these conditions, GTPase activity was enhanced by Mg2+, stimulated by mastoparan, and inhibited by GTPγS indicating the presence of either monomeric or heterotrimeric G proteins. The combination of NaF and AlCl3 is expected to inhibit heterotrimeric G protein activity but had little effect on GTPase activity in maize coleoptile membranes. Cholera toxin enhanced basal GTPase activity, confirming the presence of heterotrimeric G proteins in maize plasma membranes. Pertussis toxin also slightly enhanced basal GTPase activity in maize membranes. Abscisic acid enhanced GTPase activity optimally at 5 mmol/L Mg2+ in a concentration dependent manner by 1.5-fold at 10 µmol/L and up to three-fold at 100 µmol/L ABA. Abscisic acid induced GTPase activity was inhibited by GTPγS, the combination of NaF and AlCl3, and pertussis toxin. Overall, these results are typical of a receptor-coupled G protein responding to its ligand.

Differential Regulation of GTPase Activity by Mastoparan and Galparan

The chimeric peptide galparan, composed of galanin(1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin(1-13) was found in this tissue. EC50of 12.1 ± 2.1 μM and Hill coefficient of 2.1 ± 0.6 was calculated for galparan from a dose–response curve andKiof 19.1 ± 0.3 μM was obtained by fitting the experimental data to the Michaelis–Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose–response curve for galparan inhibition. The rate of GTPγS binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel β-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of α-helix. Critical micelle concentration of galparan in buffer solution of 22 μM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.

Phorbol ester blocks the increase of a high affinity GTPase activity induced by δ2-opioid receptor agonist in the mouse spinal cord

The high affinity GTPase activity in the mouse spinal cord was increased in a concentration-dependent manner by a selective δ 2-opioid receptor agonist, [ d-Ala 2]deltorphin II (0.1–1 μM). This increase of GTPase activity induced by [ d-Ala 2]deltorphin II was completely blocked by co-incubation with a selective δ 2-opioid receptor antagonist, naltriben (0.1 μM). A protein kinase C activator, phorbol 12,13-dibutyrate (PDB; 0.1–10 μM), which given alone had no effect on basal GTPase activity, blocked dose-dependently the increase of GTPase activity induced by [ d-Ala 2]deltorphin II (1 μM). Our results indicate the possibility that activation of protein kinase C by phorbol ester uncouples the δ 2-opioid receptor from G-proteins in the spinal cord.