Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with G beta

There is increasing evidence that heterotrimeric G-proteins (G-proteins) are involved in many plant processes including phytohormone response, pathogen defence and stomatal control. In animal systems, each of the three G-protein subunits belong to large multigene families; however, few subunits have been isolated from plants. Here we report the cloning of a second plant G-protein γ-subunit (AGG2) from Arabidopsis thaliana. The predicted AGG2 protein sequence shows 48% identity to the first identified Arabidopsis Gγ-subunit, AGG1. Furthermore, AGG2 contains all of the conserved characteristics of γ-subunits including a small size (100 amino acids, 11.1 kDa), C-terminal CAAX box and a N-terminal α-helix region capable of forming a coiled-coil interaction with the β-subunit. A strong interaction between AGG2 and both the tobacco (TGB1) and Arabidopsis (AGB1) β-subunits was observed in vivo using the yeast two-hybrid system. The strong association between AGG2 and AGB1 was confirmed in vitro. Southern and Northern analyses showed that AGG2 is a single copy gene in Arabidopsis producing two transcripts that are present in all tissues tested. The isolation of a second γ-subunit from A. thaliana indicates that plant G-proteins, like their mammalian counterparts, may form different heterotrimer combinations that presumably regulate multiple signal transduction pathways.

Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the G beta gamma dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional G alpha or G beta subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in G beta- deficient mutants while G alpha-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in G beta-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, G beta-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate- induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the G alpha- deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by G alpha. We hypothesize that G beta gamma acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole-cell patch-clamp recording techniques. VIP and PACAP (less than or equal to 10 nm) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr(1), D-Phe(2)]-GRF 1-29, amide (100 nm), a selective antagonist of VPAC(1) and VPAC(2) receptors; whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nm PACAP6-38, a PAC(1) and VPAC(2) receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 mu m GTP gamma S or GDP beta S inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTP gamma S alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alpha(o), alpha(i-1,2), alpha(i-3) or beta G-protein subunits. Only the anti-G alpha(o) and anti-G beta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive G(o) protein.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein

Background-In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Methods and Results-Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 2-1/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC50 [mol/L] NE 5.5+/-0.1, n=12; -EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC50 [mol/L] NE 5.8+/--0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC50 [mol/L] NE 5.7+/-0.2: EPI 5.8+/-0.1), Ventricular membranes from Fallot infants, labeled with (-)-[I-125]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Conclusions-Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Deletion of guanine nucleotide binding protein az subunit in mice induces a gene dose dependent tolerance to morphine

The Mechanism Underlying the development of tolerance to morphine, is still incompletely understood. Morphine binds to opioid receptors, Which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout Mouse develops significantly increased tolerance to morphine. which depends oil Galpha(z), gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved ill transducing the analgesic and lethality effects of morphine following chronic morphine treatment. (C) 2004 Elsevier Ltd. All rights reserved.

Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by G(o)-protein subunits

protein modulation of neuronal nicotinic acetylcholine receptor ( nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP gamma S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP gamma S. In inside-out membrane patches, the open probability (NPo) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2mM GTP gamma S relative to control but was unchanged in the presence of GDP gamma S. The modulation of nAChR-mediated whole- cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh- activated single-channel currents fivefold, whereas G(o)alpha( 50 nM) produced no significant increase in NPo. Neuronal nAChR subunits alpha 3-alpha 5 and alpha 2 exhibited a positive interaction with G(o)alpha and G beta gamma, whereas beta 4 and alpha 7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Control of the cystic fibrosis transmembrane conductance regulator by alpha G(i) and RGS proteins

The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown previously to be regulated by inhibitory G proteins. In the present study, we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not alphaG(0), in Xenopus oocytes. We further examined whether regulators of G protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes. Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2) partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is known to interfere with RGS proteins, counteracted the effects of RGS3. These data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because RGS proteins interfere with the G protein-dependent regulation of CFTR, this may offer new potential pathways for pharmacological intervention in cystic fibrosis. (C) 2001 Academic Press.

Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1

GCR1 has been tentatively identified in Arabidopsis thaliana as the first plant G-protein coupled receptor (GPCR) (Josefsson and Rask 1997) implicated in the cytokinin sensory pathway (Plakidou-Dymock et al. 1998). A protein fusion of GCR1 and green fluorescent protein has been expressed in Arabidopsis and shown GCR1 to be located on the plasma membrane. Studies of plants with altered GCR1 expression have led us to question GCR1's involvement in cytokinin signaling. Transgenic Arabidopsis plants containing sense and antisense constructs for GCR1 have been produced and over- and under-expression confirmed. The analysis of 12 antisense and 17 sense lines has failed to reveal the previously reported Dainty phenotype or altered cytokinin sensitivity. We have used the Gauntlet approach to test the plants' response to various plant hormones although this has not yet identified a mutant phenotype. The yeast-two hybrid system has been used and so far there is no evidence to suggest GCR1 interacts with heterotrimeric G proteins. Before GCR1 can be identified as genuine G-protein coupled receptor, the identification of a ligand and a proof of association with heterotrimeric G-proteins should be obtained.

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes, These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1), The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes, Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta -subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.