Engineering a V2 Vasopressin Receptor Agonist- and Regulator of G-Protein-Signaling-Sensitive G Protein

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.     

Effects of decavanadate and insulin enhancing vanadium compounds on glucose uptake in isolated rat adipocytes

The effects of different vanadium compounds namely pyridine-2,6-dicarboxylatedioxovanadium(V) (V5-dipic), bis(maltolato) oxovanadium(IV) (BMOV) and amavadine, and oligovanadates namely metavanadate and decavanadate were analysed on basal and insulin stimulated glucose uptake in rat adipocytes. Decavanadate (50 μM), manifest a higher increases (6-fold) on glucose uptake compared with basal, followed by BMOV (1 mM) and metavanadate (1 mM) solutions (3-fold) whereas V5 dipic and amavadine had no effect. Decavanadate (100 μM) also shows the highest insulin like activity when compared with the others compounds studied. In the presence of insulin (10 nM), only decavanadate increases (50%) the glucose uptake when compared with insulin stimulated glucose uptake whereas BMOV and metavanadate, had no effect and V5 dipic and amavadine prevent the stimulation to about half of the basal value. Decavanadate is also able to reduce or eradicate the suppressor effect caused by dexamethasone on glucose uptake at the level of the adipocytes. Altogether, vanadium compounds and oligovanadates with several structures and coordination spheres reveal different effects on glucose uptake in rat primary adipocytes.     

Stimulation of glucose transport by guanine nucleotides in permeabilized rat adipocytes.

Effects of guanine nucleotides on glucose transport were studied in permeabilized rat epididymal fat cells. GTP gamma S and Gpp(NH)p, but not App(NH)p, stimulated 3-O-methylglucose transport. Effect of GTP gamma S was dose-dependent, being detectable at 0.1 mM, and 1.0 mM GTP gamma S stimulated glucose transport to the same extent as insulin. GTP gamma S (0.3 mM) enhanced insulin-stimulated glucose transport while 1 mM GTP gamma S did not affect insulin-mediated transport. GDP beta S had no effect on glucose transport by itself but rather enhanced insulin action. NaF, which is known to activate trimeric G proteins, increased glucose transport to the same extent as insulin. Likewise, mastoparan augmented glucose transport. These results indicate that a certain type of trimeric G protein(s) is involved in the regulation of glucose transport.     

Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi

Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.     

Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins

Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding.     

Mastoparan, a wasp venom, stimulates insulin release by pancreatic islets through pertussis toxin sensitive GTP-binding protein

A wasp venom, mastoparan, rapidly stimulated insulin release by rat pancreatic islets in a dose-related manner. The amount of insulin released in response to 58 microM mastoparan far exceeded that induced by 27.8 mM glucose. Mastoparan stimulated insulin release to similar degrees at ambient glucose concentrations of 1.7 mM and 5.6 mM. The islets obtained from pertussis toxin-treated rats showed unequivocally less response to mastoparan. Pretreatment of islets with bromophenacyl bromide, a phospholipase A2 inhibitor, abolished their responsiveness to mastoparan. Pretreatment of islets with nifedipine, a Ca2+ channel blocker, was without effect. Mastoparan is a unique stimulator of insulin release by the pancreatic islets, which acts through GTP-binding protein(s) and phospholipase A2.     

The Effect of Glucose Concentration on Insulin-Induced 3T3-L1 Adipose Cell Differentiation

We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0. 1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1. 7-fold greater, and insulinstimulated phosphoinositide 3-kinase association with IRS-1 was 2. 3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses.     

Effect of insulin to decrease glucose transport in dissociated cells from the R3230AC mammary adenocarcinoma of diabetic rats.

Dissociated cells of the R3230AC mammary tumor were found to take up glucose by diffusion and by a passive carrier system. Using labeled 3-O-methylglucose as the probe, the following properties of the passive carrier were identified: (1) specificity for glucose, (2) competition by galactose and mannose but not by mannitol and fructose, (3) inhibition by phloretin but not by phloridzin, (4) temperature sensitivity, and (5) a Km for transport of 3-4 mM. The effects of insulin in vitro on carrier-mediated glucose transport were investigated in tumor cells from diabetic rats. At 10-9 M insulin, a time-related decrease in v for transport was observed resulting in an increased calculated Km (2- to 3-fold increase after 60-90 min incubation with insulin); only slight effects on V were obtained. This unusual response in v to insulin was observed when glucose was present in the medium at 2 mM and 5 mM, but not at 20 mM glucose. The effect of insulin to decrease the v was dose-related, with the major effects seen between 10-10M and 10-8M. The apparent decrease in glucose entry in vitro may in part explain the ability of insulin to inhibit growth of this tumor in vivo.     

The receptor-Galpha fusion protein as a tool for ligand screening: a model study using a nociceptin receptor-Galphai2 fusion protein

As a model system to screen endogenous ligands for G(i)-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and alpha subunit of G(i2). We detected nociceptin binding to the fusion protein by measuring stimulation of [(35)S]GTPgammaS binding with an EC(50) of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [(35)S]GTPgammaS binding to the fusion protein was clearly observed in the presence of an appropriate concentration of GDP, because the affinity for GDP was decreased in the presence of agonist. Full and partial agonists differed in their effects on apparent the affinity of the fusion protein for GDP: the IC(50) values for GDP to displace 100 pM [(35)S]GTPgammaS were estimated to be 2 micro M, 0.4 micro M, and 0.05 micro M in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [(35)S]GTPgammaS binding to the fusion protein in the brain extract derived from 2-3 g wet weight tissue without false-positive results. The active component was identified as endogenous nociceptin itself. These results indicate that the fusion protein of GPCR and Galpha(i) is useful for screening of endogenous ligands.     

Stimulation and conformational change of Goα induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oαwas highly purified from Escherichia coli cotransformed with pQE60 (G_oα) and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oαand the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that Goa·GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of Goa·GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1± 0.22)×10⁵ to (4.1±0.43)×10⁵ (M^-1). However, no obvious changes of fluorescence spectra of G_oα·GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα·GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

Alpha2-adrenergic agonist modulation of [35S]GTPgammaS binding to guanine-nucleotide-binding-proteins in human platelet membranes.

Alpha2-adrenoceptor (alpha2-AR)-regulated binding of the labelled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. Under optimal conditions, the potent alpha2-AR agonist, 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the binding of [35S]GTPgammaS up to approximately 1.8 fold, with half-maximal increase at 60 nM and was competitively inhibited by the alpha2-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated by monovalent and divalent cation levels, as well as by the concentrations of GDP. [35S]GTPgammaS binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank order of potencies of adrenergic ligands tested in stimulating [3S]GTPgammaS binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 14304> Guanabenz acetate> Oxymetazoline hydrochloride> B-HT 920 dihydrochloride> p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data presented indicate that enhancement of [35S]GTPgammaS binding by alpha2-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of ligands at the alpha2-AR.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Pertussis toxin reverses the inhibition of insulin secretion caused by [Arg8]vasopressin in rat pancreatic islets

When rat pancreatic islets were incubated with 10(-8) M arginine vasopressin in the presence of 15 mM glucose there was a pronounced inhibition of insulin release in comparison with controls. This inhibitory effect appeared to be specific for vasopressin since it was antagonised by vasopressin antibody. Moreover, pertussis toxin (100 ng/ml) reversed the inhibition of insulin release due to vasopressin, indicating the possible involvement of a guanyl-nucleotide regulatory protein in the inhibitory effect. Nevertheless, 10(-8) M vasopressin increased islet concentrations of cyclic AMP even under conditions where insulin release was decreased.     

Properties of vasopressin-activated human platelet high affinity GTPase

Effect of 8-arginine vasopressin (AVP) was examined on human platelet membrane GTPase activity as an index of a G-protein involvement. AVP stimulated a high-affinity GTPase activity in a dose-dependent manner (Kact = 1.1 +/- 0.2 nM). This stimulation was blocked by a V1a antagonist, thus confirming the V1a nature of the platelet AVP receptor. There were important variations among individuals in the AVP-induced stimulation of GTPase activity, that were in relation with the AVP-maximal binding capacity. These data suggest a causal relationship between the binding of AVP to its receptor and transduction elicited by a G-protein, without amplification. In addition, in view of the variable AVP responsiveness observed among individuals, platelet AVP-receptor appears to be subject to regulation.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Regulation of exocytosis from rat peritoneal mast cells by G protein beta gamma-subunits.

We applied G protein-derived beta gamma-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPgammaS over approximately 20-30 min. beta gamma-Subunits applied to the permeabilized cells retard this loss of sensitivity to stimulation (run-down) and it can be inferred that they interact with the regulatory mechanism for secretion. While alpha-subunits are without effect, beta gamma-subunits at concentrations >10(-8 )M enhance the secretion due to Ca2+ and GTPgammaS. Unlike the small GTPases Rac and Cdc42, beta gamma-subunits cannot induce secretion in the absence of an activating guanine nucleotide, and thus further GTP-binding proteins (likely to be Rho-related GTPases) must be involved. The enhancement due to beta gamma-subunits is mediated largely through interaction with pleckstrin homology (PH) domains. It remains manifest in the face of maximum activation by PMA and inhibition of PKC with the pseudosubstrate inhibitory peptide. Soluble peptides mimicking PH domains inhibit the secretion due to GTPgammaS and block the enhancement due to beta gamma-subunits. Our data suggest that beta gamma-subunits are components of the pathway of activation of secretion due to receptor-mimetic ligands such as mastoparan and compound 48/80.     

Nod factors activate both heterotrimeric and monomeric G-proteins in <Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.     

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding,G proteins,phosphoinositide breakdown,cyclic AMP generation,and calcium influx

Summary 1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A₁-adenosine receptors.2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTPS to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins.3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin.N-Ethylmaleimide inhibits the response to both peptides.4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTPS-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects.5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered byN-ethylmaleimide.6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.     

Regulation of I(Cl,swell) in neuroblastoma cells by G protein signaling pathways

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) activated the I(Cl,swell) anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPgammaS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPgammaS-induced current. However, cholera toxin inhibited the current approximately 70%. Exposure of cells to 8-bromoadenosine 3',5'-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPgammaS does not act through Galpha(i/o) GTPases and that Galpha(s)/Gbetagamma G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I(Cl,swell) was stimulated two- to threefold by GTPgammaS and inhibited by 10 mM guanosine 5'-O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPgammaS- and swelling-induced activation of I(Cl,swell). Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I(Cl,swell) channel via phosphorylation-independent mechanisms.