Stimulation of the release of [32P]guanosine 5'-diphosphate from G proteins in the rat anterior pituitary lobe by gonadotrophin-releasing and thyrotrophin-releasing hormones.

Plasma membranes (1-2 mg protein) purified from the anterior pituitary lobes of adult male rats were incubated with 0.6 mumol [alpha-32P]guanosine 5'-triphosphate (GTP) l-1 in an ATP-regenerating buffer at 37 degrees C for 60 min; during this incubation the [32P]GTP was hydrolysed and the nucleotide that was predominantly bound to the membranes was [32P]GDP. The release of [32P]GDP from the membranes was monitored at 37 degrees C; the amount released was proportional to the protein concentration and increased as a function of time. 5'-Guanylylimidodiphosphate (Gpp(NH)p) increased [32P]GDP release by up to 30% at 0.1 mumol l-1. Although 10 nmol Gpp(NH)p l-1 had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP-GTP exchange. Gonadotrophin-releasing hormone (GnRH) agonist and thyrotrophin-releasing hormone (TRH), at 0.1 mumol l-1 caused a maximum increase in the release of [32P]GDP of 31-38%. The GnRH agonist (0.1 mumol l-1) stimulated GDP release by 21%, 24%, 17% and 14% at 30 s, 1, 2 and 5 min, respectively. TRH (0.1 mumol l-1) stimulated GDP release by 38%, 30%, 17% and 16% at 30 s, 1, 2 and 5 min, respectively. A GnRH antagonist also stimulated [32P]GDP release, albeit less effectively than GnRH agonist; the antagonist did not inhibit agonist stimulation of GDP release. These results indicate that ligand binding to the GnRH and TRH receptors results in interaction of the receptor with a guanine-nucleotide-dependent transducer protein (G protein) and activation of GTP-GDP exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.     

Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments

The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [alpha-32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [alpha-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 microM), a specific kappa-subtype of opioid receptor agonist, inhibited the [alpha-32P]GDP release in exchange for added 1 microM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the kappa-opioid agonist at 10 microM increases the Km values of GppNHp, but not GDP in exchange for [alpha-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that kappa-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G proteins, but not by a change in affinity of GDP.     

GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.     

Adenylate cyclase activity in isolated rat islets of Langerhans Effects of agents which alter rates of insulin secretion

Adenylate cyclase activity was estimated inhomogenates of rat islets of Langerhans. by measurement of the conversion of [α-³²P]ATP to adenosine cyclic 3′,5′-[³²P]monophosphate. Islet cell adenyulate cyclase activity was stimulated by the addition to the homogenates of glucagon, fluoride, prostaglandins E₁ or E₂ GTP or CTP although not by UTP, TTP, GDP, or GMP. Adrenaline, noradrenaline and isoproterenol were each found to inhibit the activity, the order of potency at a concentration of 10^?4 M being adrenaline > noradrenaline > isoproterenol. The effects of these agents were not altered by β-blackade with propanolol but could be preventived by α-blockade with phenoxybenzamine. The following agents, present at concentrations previously shown to increase rates of insulin secretion from rat islets of Langerhans, were ineffective in altering adenylate cyclase activity when tested in the presence or absence of 0.1 mM GTP: glucose, glibenclamide, xylitol leucine, arginine, or potassium. These results suggest that the activity of adenylate cyclase in the B cells of rat islets of Langerhans may play an important role in mediating the direct effects of hormones and adrenergic agents on insulin release, although the short term effects of substrates such as glucose or amino acids on the release process do not appear to be mediated through alterations in the activity of this enzyme.     

Insulin receptor function is inhibited by guanosine 5'-[gamma-thio]triphosphate (GTP[S]).

The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [³H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [³²P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min^?1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [³²P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [³H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. ³H-agonist binding and agonist-promoted [³²P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Quantification of extracellular UDP-galactose

The human P2Y₁₄ receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y₁₄ receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with subnanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1-4-β-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [γ-³²P]ATP and the formation of [γ-³²P]UTP is monitored by high-performance liquid chromatography. The overall conversion of UDP-Gal to [γ-³²P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. The occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule.     

The roles of phospholipase D and a GTP-binding protein in guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.     

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.     

Depolarization-induced changes in the muscarinic receptor in rat brain and heart are mediated by pertussis-toxin-sensitive G-proteins

Muscarinic receptor properties in rat cortical and brain stem synaptoneurosomes and in heart myocytes were examined at resting potential and at depolarization. Depolarization induced the conversion of agonist-binding sites of the receptor from a high to a low affinity state, which could be reversed by a return to resting potential. No effect was observed on the affinity of the receptor for antagonists. Pertussis-toxin (PTX)-catalyzed ADP-ribosylation of all substrates in both synaptoneurosomal and myocyte membranes, when conducted at resting potential, prevented depolarization-induced conversion of the receptor affinity in these preparations. The target substrates were identified by [32P]ADP-ribosylation of membranes prepared from brain stem synaptoneurosomes. Autoradiography revealed labeling of a 39-kDa protein band, which reacted mainly with antibodies to the alpha-subunit of Go-proteins. The possible involvement of G-proteins in depolarization-induced changes in the receptor activity was further investigated by examining the effect of membrane potential on the PTX-sensitive binding of di- and triphosphated guanine nucleotides to synaptoneurosomal membranes. Brain stem synaptoneurosomes were made permeable to guanine nucleotides ([3H]GTP, [3H]GDP, [3H]5'-guanylyl imidodiphosphate) by treatment with ATP. After the synaptoneurosomes had been loaded with labeled GTP/GDP, resealed, and then subjected to either resting potential of short depolarization, binding of [3H]GDP to the membranes of depolarized synaptoneurosomes was 4.0 +/- 0.3 (n = 20) times higher than to the membranes of synaptoneurosomes at resting potential. Repolarization reversed this effect. Enhancement of [3H]GDP binding to the synaptoneurosomal membranes was induced also by muscarinic activation, although the increase obtained was only 30-40% (n = 5) relative to [3H]GDP binding at resting potential. Both the depolarization-induced and the muscarinically-induced enhancement of [3H]GDP binding were prevented following PTX-catalyzed ADP-ribosylation of G-proteins in the synaptoneurosomal membrane. Our results suggest that the depolarization-induced enhancement in the binding of [3H]GTP/[3H]GDP may be attributable to activation of PTX-sensitive G-proteins, which mediate the depolarization-induced alteration of the affinity of the muscarinic receptor for agonists.     

NADH Oxidase Activity of Plasma Membranes of Soybean Hypocotyls Is Activated by Guanine Nucleotides

The activity of an auxin-stimulated NADH oxidase of the plasma membrane of hypocotyls of etiolated soybean (Glycine max Merr.) seedlings responded to guanine and other nucleotides, but in a manner that differed from that of enzymes coupled to the classic trimeric and low molecular weight monomeric guanine nucleotide-binding proteins (G proteins). In the presence and absence of either auxin or divalent ions, both GTP and GDP as well as guanosine-5[prime]-O-(3-thiotriphosphate) (GTP-[gamma]-S) and other nucleoside di- and triphosphates stimulated the oxidase activity over the range 10 [mu]M to 1 mM. GTP and GTP-[gamma]-S stimulated the activity at 10 nM in the absence of added magnesium and at 1 nM in the presence of added magnesium ions. Other nucleotides stimulated at 100 nM and above. The NADH oxidase was stimulated by 10 [mu]M mastoparan and by 40 [mu]M aluminum fluoride. Neither cholera nor pertussis toxins, tested at a concentration sufficient to block mammalian G protein function, inhibited the activity. Guanosine 5[prime]-O-(2-thiodi-phosphate) (GDP-[beta]-S) did not stimulate activity, suggesting that the stimulation in response to GDP may be mediated by a plasma membrane nucleoside diphosphate kinase through conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was unaffected by nucleotides at either high or low nucleotide concentrations in the absence of added divalent ions. However, pretreatment of plasma membranes with auxin increased the apparent affinity for nucleotide binding. This increased affinity, however, appeared not to be the mechanism of auxin stimulation of the oxidase, since auxin stimulation was similar with or without low concentrations of guanine nucleotides. The stimulation by nucleotides was observed after incubating the membranes with 0.1% Triton X-100 prior to assay. The results suggest a role of guanine (and other) nucleotides in the regulation of plasma membrane NADH oxidase that differs from the interactions with G proteins commonly described for animal models.