Heterotrimeric G protein signaling in plant immunity.

In animals, heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals perceived by numerous G protein-coupled receptors (GPCRs). However, no canonical GPCRs with guanine nucleotide exchange factor (GEF) activity are present in plant genomes. Accumulated evidence indicates that, instead of GPCRs, the receptor-like kinases (RLKs) function upstream of G proteins in plants. Regulator of G protein signaling 1 (RGS1) functions to convert the GTP-bound Gα to the GDP-bound form through its GTPase-accelerating protein (GAP) activity. Because of the intrinsic differences in the biochemical properties between Arabidopsis and animal Gα, the actions of animal and Arabidopsis RGS1 result in contrasting outcomes in G signaling activation/deactivation. Animal RGSs accelerate the deactivation of the activated G signaling, whereas Arabidopsis RGS1 prevents the activation of G signaling in the resting state. Phosphorylation of Arabidopsis RGS1 triggered by ligand-RLK recognition results in the endocytosis or degradation of RGS1, leading to the separation of RGS1 from Gα and thus the derepression of G signaling. Here, we summarize the involvement of the G proteins in plant immunity, with a special focus on the molecular mechanism of G signaling activation/deactivation regulated by RLKs and RGS1. We also provide a brief perspective on the outstanding questions that need to be addressed to fully understand G signaling in plant immunity.

Regulation of immune function by G protein-coupled receptors, trimeric G proteins, and RGS proteins.

Receptors for chemokines and a variety of ligands such as histamine, nucleosides, and bioactive lipids signal through heterotrimeric G proteins and play critical roles in immune function. Heterotrimeric G protein signaling pathways are subjected to many layers of regulation including regulators of G protein signaling (RGS) proteins that mainly function to attenuate these signaling pathways. This review focuses on the overall importance of G protein-coupled receptor-heterotrimeric G protein-RGS protein signaling in immune function with emphasis on lymphocyte trafficking and motility. Considerable portion is devoted to discussing mechanisms by which chemoattractant receptors activate downstream signaling pathways that function during leukocyte migration. Studies using intravital imaging techniques to monitor lymphocyte trafficking and motility as well as ones probing intracellular spatiotemporal dynamics of trimeric signaling components are also discussed as they increasingly provide mechanistic insights into trimeric G protein signaling networks.

Papel de las quimiocinas en el desarrollo de los linfocitos T / Role of Chemokines in T cell development

Las quimocinas juegan un papel muy importante en el desarrollo tímico y la migración específica a órganos linfoides. Las quimiocinas son citocinas quimioatrayentes de bajo peso molecular y ejercen su función al interaccionar con receptores de siete dominios transmembranales asociados a proteínas G heterotriméricas. Como consecuencia de la unión de la quimiocina con su receptor se activa la vía Jak/Stat, que parece ser crucial para la generación de respuestas funcionales tales como la quimiotaxis y la movilización de calcio intracelular. La expresión diferencial de quimiocinas y sus receptores durante los distintos estadíos de diferenciación de los timocitos permite la migración dirigida de timocitos inmaduros desde la región subcapsular hasta la médula, pasando a través de la corteza. Además, existen evidencias de que las quimocinas también están implicadas en el reclutamiento de progenitores hasta el timo así como en la salida de los timocitos maduros hacia órganos linfoides secundarios. La generación de ratones deficientes para quimiocinas y/o sus receptores ha sugerido que ciertas quimiocinas pueden tener un papel redundante, mientras que otras dan lugar a un fenotipo muy definido, como el caso del receptor CXCR4 y su ligando CXCL12 cuya deficiencia resulta letal a nivel embrionario (AU)

G protein-coupled receptors in natural killer cells.

Natural killer (NK) cells are capable of killing tumor as well as virally infected cells. How these cells migrate toward the infected sites in the body is not completely understood. Chemokine receptors that belong to the heptahelical family of receptors and characteristically bind heterotrimeric G proteins are present in most NK cells. Recent results showed that resting NK cells highly express constitutive chemokine receptors (CCR4, CCR7, CXCR4, and CX(3)CR1) with low expression of a limited repertoire of inflammatory chemokine receptors (CCR1 and CXCR3). However, only a subset of these cells expressing the CD56(dim) and adhesion molecule(high) phenotype is capable of in vivo binding to vascular endothelium. Under pathological conditions where inflammatory cytokines are present, these cells are induced to express inflammatory chemokine receptors. Resting as well as activated NK cells also express receptors for another member of the heptahelical family of receptors that bind phosphorylated or glycosylated lysolipids. These include sphingosine 1-phosphate (S1P)(1), S1P(4), and S1P(5), the receptors for S1P; lysophosphatidic acid (LPA)(1), LPA(2), and LPA(3), the receptors for LPA; and T cell death-associated gene 8, the receptor for psychosine. Similar to chemokines, S1P, LPA, and psychosine induce the chemotaxis of NK cells through heterotrimeric G proteins. However, in contrast to chemokines, which enhance the cytotoxicity of NK cells, lysolipids inhibit this function. We hope that gaining knowledge regarding the distribution of activated NK cells toward the sites of tumor growth or virally infected sites will give an advantage in designing strategies using these cells as tools for the prevention and treatment of immunodeficiencies.

Mutational uncoupling of alpha1A-adrenergic receptors from G proteins also uncouples mitogenic and transcriptional responses in PC12 cells.

Activation of human alpha1A-adrenergic receptors in PC12 cells causes many second messenger, mitogenic, and transcriptional responses. We examined the role of G protein activation in these responses by uncoupling the receptor through deletion of the first three amino acids from the third intracellular loop (Delta208-210). Expression levels of retrovirus-transfected wild-type and Delta(208-210) alpha1A-adrenergic receptors in PC12 cells were similar and showed identical binding affinities for antagonists. However, the potency of the agonist norepinephrine was increased 9-fold by the Delta (208-210) mutation. In PC12 cells expressing the Delta (208-210) construct, calcium and inositol phosphate responses to norepinephrine were essentially abolished. The strong activation of mitogen-activated protein kinase pathways seen upon stimulation of wild-type alpha1A-adrenergic receptors in PC12 cells was abolished by the Delta (208-210) mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through alpha1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-kappaB, nuclear factor of activated T cells, gamma-interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the Delta (208-210) mutation. Overexpression of Galpha16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of alpha1A-adrenergic receptors in PC12 cells all require G protein activation.

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach.

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.

Galpha12- and Galpha13-protein subunit linkage of D5 dopamine receptors in the nephron.

The roles of the G-protein alpha-subunits, Gs, Gi, and Gq/11, in the signal transduction of the D1-like dopamine receptors, D1 and D5, have been deciphered. Galpha12 and Galpha13, members of the 4th family of G protein subunits, are not linked with D1 receptors, and their linkage to D5 receptors is not known. Therefore, we studied the expression of Galpha12 and Galpha13 and interaction with D5 dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D5 receptor-transfected HEK293 cells. Galpha12 and Galpha13 were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas Galpha12 was expressed in the ascending limb of Henle, Galpha13 was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, Galpha12 and Galpha13, as with D5 receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D5 receptors with Galpha12 and Galpha13 in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D5 receptor-transfected HEK293 cells. In these cells, a D1-like agonist, fenoldopam, increased the association of Galpha12 and Galpha13 with D5 receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D1 and D5 receptors are linked to Galphas, they are differentially linked to Galpha12 and Galpha13. The consequences of the differential G-protein subunit linkage on D1- and D5-mediated sodium transport remains to be determined.

Raft.1, a monoclonal antibody raised against the raft microdomain, recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line.

SUMMARY: Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.

Antibody capture assay reveals bell-shaped concentration-response isotherms for h5-HT(1A) receptor-mediated Galpha(i3) activation: conformational selection by high-efficacy agonists, and relationship to trafficking of receptor signaling.

Although serotonin 5-HT(1A) receptors couple to several Gi/o G-protein subtypes, little is known concerning their differential activation patterns. In this study, in membranes of Chinese hamster ovary cells expressing h5-hydroxytryptamine(1A) receptors (CHO-h5-HT(1A)), isotherms of 5-HT-stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Galpha(i3) subunits and was resistant to induction of [(35)S]GTPgammaS dissociation by unlabeled GTPgammaS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Galpha(i3) activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [(35)S]GTPgammaS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Galpha(i3) stimulation progressively returned to basal values. In contrast, the partial agonists (-)-pindolol and 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) displayed sigmoidal stimulation isotherms, whereas spiperone and other inverse agonists sigmoidally inhibited [(35)S]GTPgammaS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Galpha(i3) activation were i) abolished by pretreatment of CHO-h5-HT(1A) cells with pertussis toxin; ii) reversed by the selective 5-HT(1A) antagonist (N-[2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclohexane-carboxamide) fumarate (WAY100,635), and iii) absent in nontransfected CHO cell membranes. 5-HT isotherms could be modified by altering sodium concentration; only stimulatory actions were observed at 300mM NaCl, whereas only inhibitory actions were seen at 10 mM NaCl. Furthermore, bell-shaped isotherms were not detected at short incubation times, suggesting time-dependent changes in receptor/Galpha(i3) coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT(1A) agonists direct receptor signaling to Galpha(i3). In contrast, partial agonists favor h5-HT(1A) receptor signaling to Galpha(i3) over a wide concentration range, whereas inverse agonists inhibit constitutive Galpha(i3) activation.

Elevated nasal mucosal G protein levels and histamine receptor affinity in a guinea pig model of nasal hyperresponsiveness.

BACKGROUND: Nasal hyperresponsiveness is a common feature of allergic rhinitis, but the underlying mechanisms have yet to be elucidated. The effects of repeated antigen inhalation on the characteristics of histamine H(1) receptors and expression levels of heterotrimeric guanosine 5'-triphosphate-binding proteins in nasal mucosa were investigated to understand the mechanisms of the pathogenesis of nasal hyperresponsiveness in allergic rhinitis. METHODS: Male Hartley guinea pigs were sensitized by the inhalation of dinitrophenylated ovalbumin antigen (10 mg of protein/ml) and repeatedly challenged by inhaling aerosolized dinitrophenylated ovalbumin antigen for 3 weeks. Twenty-four hours after the last antigen inhalation, in vivo nasal responsiveness to histamine was measured. [(3)H]Mepyramine binding assays and immunoblotting for alpha subunits of the G(q) protein were also performed using membrane preparations of isolated nasal mucosae. RESULTS: The histamine-induced increase in intranasal pressure was significantly augmented after repeated antigen challenge, indicating that nasal hyperresponsiveness was achieved. In saturation binding studies, no significant change was observed in the density and antagonist affinity of H(1) receptors in the hyperresponsive animals. On the other hand, the affinity of histamine for high-affinity agonist binding sites in the hyperresponsive group, measured by histamine competition binding studies, was much greater than that in control animals, and these results were affected by guanosine 5'-O-(3-thiotriphosphate) in both groups. Moreover, Galpha(q) levels in nasal mucosal homogenates were significantly increased after repeated antigen challenge. CONCLUSIONS: Elevated G protein levels in nasal mucosa might induce an increased binding affinity of histamine to its receptors, resulting in an augmented nasal response to histamine, that is, nasal hyperresponsiveness, in guinea pigs.

G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla.

G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.

A novel site on the Galpha -protein that recognizes heptahelical receptors.

Specific domains of the G-protein alpha subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between alpha4 and alpha5 helices of the G-protein alpha subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Galpha(15) and its human homologue Galpha(16), whereas metabotropic glutamate receptor 8 activated Galpha(15) only. The extreme C-terminal 20 amino acid residues are identical between the Galpha(15) and Galpha(16) and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Galpha(16) that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Galpha(16). One module comprises alpha4 helix, alpha4-beta6 loop (L9 Loop), beta6 sheet, and alpha5 helix. The other, not described previously, is located within the loop that links the N-terminal alpha helix to the beta1 strand of the Ras-like domain of the alpha subunit. Coupling of Galpha(16) protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.

Distinct guanine nucleotide binding protein alpha-subunit receptor coupling in GH cell lines: effects of bromocriptine and hormones on effector enzyme modulation.

The purpose of the present study was to elucidate how the dopamine agonist bromocriptine affected receptor-effector systems in GH cells by measuring adenylate cyclase (AC) and phospholipase C (PL-C) modulation in cell membrane preparations. To perturb the interaction between the receptor and G-protein, polyclonal antibodies reacting with the predicted C-terminal amino acid sequence of G-protein alpha-subunits were used. The effect of bromocriptine on secretagogue elicited prolactin (PRL) secretion from whole cells was also monitored. Bromocriptine inhibited the basal secretion of PRL in a dose dependent manner, and completely abolished both the thyroliberin (TRH) and the vasoactive intestinal peptide (VIP) stimulated PRL secretion in GH(3) cells. Maximal inhibitory effect on PRL egress elicited by both hormones was obtained at 10-50 microM of bromocriptine. Messenger RNAs for both the short and long form of the D(2) receptor (D(2)R) were demonstrated in all three GH cell lines using the RT-PCR technique, advocating that D(2)Rs are coupled to distinct G-proteins and, thus, probably being responsible for the observed effects of bromocriptine in these cell lines. Basal AC activity, as measured in membrane preparations of GH(3) cells, remained unaffected by bromocriptine treatment (10 microM), while TRH and VIP stimulated AC activities (175% and 350% of control values, respectively) were partially inhibited (by some 50%). This inhibitory effect of bromocriptine was completely and specifically abolished in the presence of an antiserum against G(i2)alpha. Basal PL-C activity was also unaffected by bromocriptine, while TRH stimulated PL-C activity (350% of control value) was inhibited by bromocriptine (10 microM) by approximately 50%. Immunoblocking of G(q/11)alpha, however, reduced the stimulatory effect of TRH on PL-C activation by some 65%, while an antiserum against G(o)alpha partly counteracted the inhibitory effect of bromocriptine (10 microM) on TRH stimulated PL-C activity. Thus, TRH dependent AC stimulation was counteracted by bromocriptine via G(i2). TRH activation of PL-C occurs via G(q/11), while inhibition by bromocriptine appears to involve G(o). These mechanisms probably account for the major part of the actions of bromocriptine, however, other not yet recognised intermediates may be involved.

[A case report of Bickerstaff's brainstem encephalitis with positive anti GQ 1 b, GT 1 a, GM 1 ganglioside antibodies].

Patient was an 18-year-old female student. After she had symptoms of common cold for 3 days, she developed somnolence, diplopia, dysarthria, urinary disturbance and ataxia. On admission neurological examination revealed coma with mydriasis, ophthalmoplegia, ptosis and weakness of the upper limbs. Light reflex, corneal reflex and oculocephalic test were all negative. Deep tendon reflexes were brisk and extensor toe signs were positive bilaterally. She did not have nuchal rigidity. Laboratory test revealed normal cerebrospinal fluid with negative myelin basic protein. Brain MRI, brainstem evoked potentials presented no abnormality. EMG revealed normal conduction velocity and no conduction block. EEG had diffuse theta and delta slowing. Culture of the stool represented no Campylobacter jejuni. At the fifth day of admission consciousness level improved, and other neurological findings disappeared in about 6 weeks. She had anti GQ 1 b, GT 1 a(IgG, IgM) and anti GM 1(IgM) antibodies in the serum. We made a diagnosis of Bickerstaff's brainstem encephalitis from these neurological symptoms and clinical course. The main lesion was present in the brainstem from midbrain to medulla oblongata in the midline. High titer of anti GT 1a antibody may be related to the ophthalmoplegia as noted in Miller Fisher syndrome. As a result of EMG and stool culture, it denied the complication of Guillain-Barré syndrome. We had no proof of the reason of the presentation of anti GM 1 antibody.

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 (

Heterotrimeric G-protein beta-subunit is localized in the plasma membrane and nuclei of tobacco leaves.

Heterotrimeric G-proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plants. Antibodies raised against the tobacco heterotrimeric G-protein beta-subunit (Gbeta) were used to analyse its distribution in tobacco leaves. In young tissue the protein level was relatively high, while it declined substantially during later stages of leaf development. Cell fractionation revealed that Gbeta is tightly associated with plasma membrane, but can also be detected in purified nuclei.

Somatotropin has no effect on the quantity of guanine nucleotide binding proteins Gqalpha/G11alpha in goat adipose tissue in vivo.

The decapeptide QLNLKEYNLV corresponding to the C-terminus of Gq/G11alpha guanine nucleotide-binding proteins (G-proteins) was synthesized by the solid-phase method and conjugated to keyhole limpet hemocyanin. The rabbits were immunized with these conjugates and an antiserum that reacted specifically with the alpha subunit of Gq/G11 proteins was used in this study. The antiserum exhibited no cross-reactivity with the alpha subunits of stimulatory (Gs) or inhibitory (Gi) G-proteins associated with adenylate cyclase. Immunoblots with the antiserum showed that it specifically recognized the Gq/G11 alpha-proteins in cholate extracts of adipose tissue membranes of goats. Treatment of young castrated male goats with bST had no effect on the quantity of Gq/G11 alpha subunits in adipose tissue and the results thus obtained did not support the idea that the bST signal in adipose tissue is transmitted via Gq/G11 alpha-proteins.