Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.     

Expression of human muscarinic cholinergic receptors in tobacco

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0–2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.     

Cross Talk between β-Adrenergic and Bradykinin B2 Receptors Results in Cooperative Regulation of Cyclic AMP Accumulation and Mitogen-Activated Protein Kinase Activity

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.     

Quantitative and qualitative differences in guanine nucleotide binding protein activation by formyl peptide and leukotriene B4 receptors.

Formyl peptides and leukotriene B4 (LTB4) stimulate disparate neutrophil functional responses and second messenger generation. The hypothesis that differences in receptor-guanine nucleotide-binding proteins (G protein) interaction account for the disparate responses was examined using HL-60 granulocyte plasma membranes. The quantity of receptor-coupled G proteins was determined by guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) equilibrium binding in the presence or absence of f-Met-Leu-Phe and/or LTB4. About one-third of the total GTP gamma S binding sites were coupled to f-Met-Leu-Phe receptors, to LTB4 receptors, and to receptors when both ligands were added simultaneously. The dissociation constant of GTP gamma S-binding sites in the presence of LTB4 was significantly greater than that in the presence of f-Met-Leu-Phe. f-Met-Leu-Phe shifted the GDP dose-inhibition curve for GTP gamma S binding further to the right than did LTB4. The apparent initial rate of GTP hydrolysis and GTP gamma S binding stimulated by f-Met-Leu-Phe was significantly greater than that stimulated by LTB4. There were significantly more formyl peptide receptors than LTB4 receptors, however, formyl peptide and LTB4 receptor density did not differ under GTP gamma S binding assay conditions. The rate of GTP hydrolysis stimulated by LTB4 was not increased in membranes containing twice the LTB4 receptor density. We conclude that formyl peptide receptors stimulate more rapid activation of a common pool of G proteins than LTB4 receptors because of a significantly reduced affinity of formyl peptide receptor-activated G proteins for GDP.     

Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles.

The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli. Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma. Efficient regulation of alpha subunit by receptor required the presence of beta gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.     

Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.     

Guanine nucleotides regulate beta-adrenergic activation of Na-H exchange independently of receptor coupling to Gs.

We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.     

Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.     

Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity

beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.     

An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism.

G protein-coupled membrane receptors activate G proteins by enhancing guanine nucleotide exchange. G0 is a major component of the growing regions (growth cones) of neurons. GAP-43 is a neuronal protein associated with the cytosolic face of the growth cone plasma membrane and stimulates binding of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to Go (Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Nature 344, 836-841). Here we have examined the mechanism by which GAP-43 affects G0. Like G protein-coupled receptors, GAP-43 enhances GDP release from G0, increases the initial rate of GTP gamma S binding, and increases the GTPase activity of Go, all without altering the intrinsic kappa cat for the GTPase. Unlike the case for receptors, however, the GAP-43 effect is not blocked by pertussis toxin, nor affected by the presence or absence of beta gamma or of phospholipids. There is specificity to the interaction, in that GAP-43 increases GTP gamma S binding to recombinant alpha o and alpha i1, but not to recombinant alpha s. Thus, GAP-43 is a guanine nucleotide release protein with a novel mechanism of action, potentially controlling membrane-associated G proteins from within the cell.     

Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C

The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Molecular mechanisms of regulatory action of adrenergic receptor agonists on functional activity of adenylyl cyclase signaling system of the ciliates Dileptus anser and Tetrahymena pyriformis

Adenylyl cyclase signaling system (ACS) of the higher eukaryotes involves the following main components: receptor, heterotrimeric G protein, adenylyl cyclase (AC), and protein kinase A. At present, these components have been found in cells of different species of the lower eukaryotes. Hence, the signal transduction through ACS of unicellular eukaryotes may have some features in common with those of the higher eukaryotes. We showed earlier that agonists of adrenergic receptors (ARs) regulate AC activity of ciliates Dileptus anser and Tetrahymena pyriformis. The aim of this work was to study molecular mechanisms of AR ligand action on the functional activity of different components of ACS of the ciliates. It has been shown that beta-AR antagonist [3H]-dihydroalprenolol binds membranes of the ciliates with a comparatively lower affinity than those of the higher eukaryotes (Kd for D. anser was 13.4 nM, for T. pyriformis--27 nM). Beta-AR ligands--agonist (-)-isoproterenol and antagonists propranolol and atenolol in competition manner displace [3H]-dihydroalprenolol with IC50 that are 10-100 times higher than corresponding IC50 of beta-AR of the higher eukaryotes. In the presence of GTP, the right shift of competition curves of [3H]-dihydroalprenolol displacement by isoproterenol was obtained, being most considerable in the case of D. anser. Adrenaline and isoproterenol in a dose-dependent manner stimulated GTP-binding in cell cultures of D. anser and T. pyriformis. Suramin (10(-5) M), the inhibitor of heterotrimeric G proteins, completely blocked effects of these hormones. In D. anser culture, adrenaline and isoproterenol in a dose-dependent manner, stimulated AC activity, and its stimulating effects in the presence of beta-AR blockers vanished (propranolol) or decreased to a great extent (atenolol). At the same time the effects were unchanged in the presence of alpha2-AR antagonists yohimbine and idazoxan. These data show the involvement of G protein-coupled beta-AR in signal transduction induced by AR agonists in D. anser cells. In cell culture of T. pyriformis isoproterenol weakly stimulated AC activity, and its effect was completely blocked by beta-AR blockers. Adrenaline in T. pyriformis cells in a dose-dependent manner inhibited AC activity. Inhibiting effect of hormone was decreased in the presence of alpha2-AR blockers. On the basis of the obtained data we concluded that adrenaline in T. pyriformis cells inhibited AC activity through G protein-coupled receptor, being close to alpha2-AR of vertebrate animals.     

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.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Intracellular signaling events at the leading edge of migrating cells

Cell migration is an important facet of the life cycle of immune and other cell types. A complex set of events must take place at the leading edge of motile cells before these cells can migrate. Chemokines induce the motility of various cell types by activating multiple intracellular signaling pathways. These include the activation of chemokine receptors, which are coupled to the heterotrimeric G proteins. The release of G beta gamma subunits from chemokine receptors results in the recruitment to the plasma membrane, with subsequent activation of various down-stream signaling molecules. Among these molecules are the pleckstrin homology domain-containing proteins and the phosphoinositide 3-kinase gamma which phosphorylates phospholipids and activates members of the GTP exchange factors (GEFs). These GEFs facilitate the exchange of GTP for GDP in members of GTPases. The latter are important for reorganizing the cell cytoskeleton, and in inducing chemotaxis. Chemokines also induce the mobilization of intracellular calcium from intracellular stores. Second messengers such as inositol 1,4,5 trisphosphate, and cyclic adenosine diphosphate ribose are among those induced by chemokines. In addition, the G beta gamma subunits recruit members of the G protein-coupled receptor kinases, which phosphorylate chemokine receptors, resulting in desensitization and termination of the motility signals. This review will discuss the intracellular signaling pathways induced by chemokines, particularly those activated at the leading edge of migrating cells which lead to cell polarization, cytoskeleton reorganization and motility.