Lysophosphatidic acid inhibits the cytotoxic activity of NK cells: involvement of Gs protein-mediated signaling.

Lysophosphatidic acid (LPA) is an activator and chemoattractant of NK cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of LPA on the interaction of human NK cells with tumor cells such as the Burkitt lymphoma cell line Raji and the human melanoma cell line A2058. Thereby we found that LPA inhibits the release of perforin and cytotoxic activity of NK cells. Analysis of signal transduction showed that LPA induces common signaling pathways of chemotaxins such as G(i) protein-dependent actin re-organization, activation of the mitogen-activated protein kinase p38 as well as phosphatidylinositol-3-kinase-dependent signal molecules [protein kinase B/Akt and glycogen synthase kinase-3beta (GSK-3beta)]. In contrast to most chemotaxins, LPA is also able to activate G(s)-dependent signaling molecules. This signaling cascade involves the LPA receptor type-2, increase cAMP levels and protein kinase A (PKA) activation, which in turn are responsible for the modulatory effect of LPA on NK cell-mediated cytotoxicity. Moreover, blocking the regulatory subunits of PKA I abrogates the inhibitory effect of LPA, whereas the catalytic subunits are not involved. Based on our data, one can assume that LPA contributes to the tumor escape from the immunological surveillance machinery.

Meiotic arrest in human oocytes is maintained by a Gs signaling pathway.

In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.

LC MALDI-TOF MS/MS and LC ESI FTMS analyses of HLA-B27 associated peptides isolated from peripheral blood cells.

Peptides eluted from peripheral blood cells of HLA-B*2705 healthy donor were analyzed by LC MALDI MS/MS and LC ESI FTMS techniques. The sequences of 92 peptide ligands identified from one healthy blood donor by LC MALDI-TOF MS/MS were compared with those previously published from in vitro long-term cell cultures available in SYFPEITHI database and splenocytes. It was found that 18 sequences confirmed within 1ppm mass error by LC ESI FTMS were already described and 3 of them matched with those previously reported from HLA-B*2705 splenocytes. Another 38 sequences validated within the same mass error were not found in SYFPEITHI database and are identified here for the first time. Finally, 36 sequences (5 sequences already published in SYFPEITHI database) were evaluated by LC MALDI-TOF MS/MS but no matches in the list of monoisotopic masses obtained from LC ESI FTMS were found.

Meiotic resumption of porcine immature oocytes is prevented by ooplasmic Gsalpha functions.

A high cyclic adenosine monophosphate (cAMP) level in fully-grown immature oocytes prevents meiotic resumption. In Xenopus, inhibitory cAMP is synthesized within oocytes depending on a stimulatory alpha-subunit of G-protein (Gsalpha). In the present study, we examined whether ooplasmic Gsalpha is involved in meiotic arrest of porcine oocytes. First, we studied the presence of Gsalpha molecules in porcine oocytes by immunoblotting, and this suggested the presence of reported isoforms (45 and 48 kDa) not only in cumulus cells but also in porcine oocytes. Then we injected an anti-Gsalpha antibody into porcine immature oocytes and found that inhibition of ooplasmic Gsalpha functions significantly promoted germinal vesicle breakdown of the oocytes, whose spontaneous meiotic resumption was prevented by 3-isobutyl-l-methylxanthine (IBMX) treatment. Although cyclin B synthesis and M-phase promoting factor (MPF) activation were largely prevented until 30 h of culture in IBMX-treated oocytes, injection of anti-Gsalpha antibody into these oocytes partially recovered cyclin B synthesis and activated MPF activity at 30 h. These results suggest that meiotic resumption of porcine oocytes is prevented by ooplasmic Gsalpha, which may stimulate cAMP synthesis within porcine oocytes, and that synthesized cAMP prevents meiotic resumption of oocytes through the signaling pathways involved in MPF activation.

Identification of HLA-CW3, GNAS and IMPA as cytotoxic T-lymphocyte (CTL) target antigens using an allogeneic mixed lymphocyte tumor cell culture (MLTC) system and subsequent cDNA library screening.

Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established using lymphocytes from non-small-cell lung cancer (NSCLC) patient UKY-53 and HLA-A2+ NSCLC tumor cells (UKY-29). The tumor cells expressed the lymphocyte costimulatory molecule CD80 (UKY29.7). Cytolytic activity showed the cytotoxic T-lymphocytes (CTL) lysed UKY-29, but not K562 or Daudi. The CTL also lysed: HLA-A2+ and -A24+ tumor cell lines from a number of tumor histologies. The CTL also lysed Epstein Barr virus transformed (EBV) B-cells, UKY-29EBV, autologous to the stimulating cell line, UKY29TC. These data suggested the presence of both tumor-specific and allogeneic reactivities in the bulk CTL population. Subsequent cDNA cloning analysis and sequencing demonstrated that the bulk CTL population was recognizing: (i) allogeneic target HLA-CW3, and two minor histocompatibility antigens; (ii) guanine nucleotide-binding protein, G(S) (GNAS), and (iii) inositol myophosphatase (IMPA). All three antigens, we believe, were restricted by HLA-A2. Whereas the system described was initially intended to identify tumor-associated antigens recognized by CTL, the nature of the allogeneic system provides a unique opportunity for the identification of epitopes that confer both allo and minor antigen recognition.

Dopamine D1 receptor coupling to Gs/olf and Gq in rat striatum and cortex: a scintillation proximity assay (SPA)/antibody-capture characterization of benzazepine agonists.

Cloned, human dopamine D(1) receptors recruit multiple effectors but the G-protein subtype(s) activated by cerebral populations remain poorly defined, a question addressed using a rapid immunocapture technique. In rat striatum, dopamine (DA) and four selective, benzazepine agonists at D(1) receptors concentration-dependently enhanced [(35)S]GTPgammaS binding to Galphas/olf. For all drugs, Galphaq was also recruited with similar potencies and efficacies. Comparable observations were made in the cortex wherein profiles of Galphas/olf vs Galphaq activation were also highly correlated. In contrast to Galphas/olf and Galphaq, Galphao and Galphai were activated neither in the striatum nor in the cortex, except for SKF82958. As compared to DA, both SKF81297 and SKF82958 were full agonists at Gs/olf and Gq in cortex and striatum, whereas SKF38393 behaved as a partial agonist. Likewise, the "atypical" agonist, SKF83959 only partially activated Galphaq and also Gs/olf in these two regions. In both striatum and cortex, the selective D(1) receptor antagonist, SCH23390, abolished the recruitment of Galphaq and Galphas by DA, and the action of DA was partially attenuated by SKF83959. These findings demonstrate that, in native CNS tissue, DA and other D(1) receptor agonists activate Galphas and Galphaq with similar potencies and efficacies, suggesting their recruitment via pharmacologically-indistinguishable populations of D(1) receptors, and show that SPA technology is well-adapted to study the coupling of native DA receptors.

Lack of mutations in the TSHr and Gsalpha genes in TSHr antibody negative Graves' disease.

The aim of this study was to investigate whether TSHr antibody negative Graves' disease is associated with somatic mutations in the TSHr or Gsalpha genes and whether histopathologically defined thyroid lesions, i.e., hyperfunctioning adenoma, non-functioning follicular adenomas, or nodules in toxic and non-toxic multinodular goiters are associated with such mutations. No mutations but three germ-line polymorphisms were found in patients with TSHr antibody negative Graves' disease. The three polymorphisms are expected to have no or only minor effects on the signaling properties, and is not associated with altered antigenecity imposed by such mutations. Two heterozygous somatic TSHr mutations were found in two hyperfunctioning adenomas and in two toxic multinodular goiters. The lack of TSHr and Gsalpha mutations in TSHr antibody negative Graves' disease patients indicates that such mutations are neither primary nor secondary events in this disease. The results also confirm that somatic gain-of-function TSHr mutations are present in hyperfunctioning follicular adenomas and goiters, but not in non-functioning thyroid lesions.

Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine.

Herein, we examined the direct coupling of human dopamine D1 receptors to G(s) proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists ( approximately 100%) in stimulating guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding to G(s) in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTP gamma S binding to G(s) with a pK(B) (9.29) close to its pK(i) (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G(s) activation with pK(B)'s of 6.7 and 7.3, respectively, values close to their pK(i) values at these sites. In conclusion, the use of an anti-G(s) protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.

Meiotic arrest in the mouse follicle maintained by a Gs protein in the oocyte.

The mammalian ovarian follicle consists of a multilayered complex of somatic cells that surround the oocyte. A signal from the follicle cells keeps the oocyte cell cycle arrested at prophase of meiosis I until luteinizing hormone from the pituitary acts on the follicle cells to release the arrest, causing meiosis to continue. Here we show that meiotic arrest can be released in mice by microinjecting the oocyte within the follicle with an antibody that inhibits the stimulatory heterotrimeric GTP-binding protein Gs. This indicates that Gs activity in the oocyte is required to maintain meiotic arrest within the ovarian follicle and suggests that the follicle may keep the cell cycle arrested by activating Gs.

Persistent activation of Gsalpha through limited proteolysis by calpain.

Treatment of rat pituitary GH(4)C(1) cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but did not attenuate MnCl(2) action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of G(s)alpha, but not by anti-G(i)2alpha or anti-Gbeta antibodies. Furthermore, it was shown that G(s)alpha is more susceptible to calpain-mediated proteolysis than G(i)2alpha or Gbeta. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of G(s)alpha in GH(4)C(1) cell membranes. Proteolysis of G(s)alpha by micro-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH(4)C(1) cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH(4)C(1) cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH(4)C(1) cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of G(s)alpha, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of G(s)alpha to an active G(s)alpha fragment. This is a novel calpain-dependent activation mechanism of G(s)alpha and, thus, of adenylate cyclase in rat pituitary cells.

An antibody directed against residues 100-119 within the alpha-helical domain of Galpha(s) defines a novel contact site for beta-adrenergic receptors.

A polyclonal antiserum that recognizes residues 100-119 within the alpha-helical domain of Galpha(s) (K-20) caused a dissociation of G(s) into its component subunits and activated a cholera toxin-sensitive high affinity GTPase. Consistently, the antibody mimicked the stimulatory effects of the beta-adrenergic agonist, isoproterenol, on adenylyl cyclase, which is mediated by Galpha(s), and its inhibitory action on NADPH-dependent H(2)O(2) generation, a Gbetagamma-mediated response. A peptide corresponding to the target sequence of K-20 not only neutralized the receptor-mimetic effects of the antibody but inhibited the whole spectrum of isoproterenol action as well, including its antagonistic effects on adenylyl cyclase and NADPH-dependent H(2)O(2) generation. By contrast, COOH-terminal anti-Galpha(s) selectively inhibited the stimulatory effect of isoproterenol on cAMP formation without affecting its inhibitory effect on NADPH-dependent H(2)O(2) generation. The data are consistent with the concept that beta-adrenergic receptors interact with multiple sites on Galpha(s) each playing a distinct role, and strongly suggest that antibody K-20 defines a novel contact site for beta-adrenergic receptors that localizes to the alpha-helical domain and is essential for eliciting the complete spectrum of beta-adrenergic responses.

Entamoeba histolytica: identification of functional Gs and Gi proteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.

Trophozoites of Entamoeba histolytica adhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP3, Ca2-, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated by Vibrio cholerae and Bordetella pertussis toxins. Three of them are also recognized by antibodies prepared against the alpha-subunit of Gs-and Gi-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of Gs- and Gi-like proteins and their apparent activation during in vitro interaction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms in E. histolytica.

Thyroid status affects the rat cardiac beta-adrenoceptor system transiently and time-dependently.

1. The aim of this study was to investigate the time-dependency of the influence of dysthyroid states on the beta-adrenoceptor system in rat heart left ventricle. Therefore, the influence of acute and chronic hyper- and hypothyroidism on beta-adrenoceptor-induced left ventricular responses, beta-adrenoceptor density, cardiac noradrenaline tissue concentrations, Gs alpha-proteins, and basal and stimulated adenylate cyclase activities was determined. 2. Hyperthyroid rats were obtained by feeding with thyroxine (T4)-containing rat-chow for 1, 4 and 8 weeks. Hypothyroidism was induced by adding 0.05% propylthiouracil (PTU) to the drinking water. Rats of varying ages were used in order to compensate for the differences in the duration of the treatments. Rats were aged 3 and 5 months at the end of the experiments. 3. Thyroxine treatment for 4 and 8 weeks increased the cardiac sensitivity to isoprenaline, but maximal induced inotropic responses were decreased. Cardiac ventricular beta-adrenoceptor density was increased only in rats treated with T4 for 1 week. This transient effect of hyperthyroidism on cardiac beta-adrenoceptor density was not observed in older rats. The PTU treatment resulted in a stable decrease of cardiac beta-adrenoceptor density. 4. Left ventricular tissue noradrenaline concentrations were unaffected by hyperthyroidism, where a decrease was observed in hypothyroid rats. Density of Gs alpha proteins was increased in hearts from chronic hyperthyroid rats. 5. These results indicate that the increased sensitivity to beta-adrenoceptor-mediated stimulation in chronic hyperthyroidism cannot be attributed to changes in cardiac beta-adrenoceptor density, but is probably caused by an enhanced content of Gs alpha. Accordingly, in hyperthyroidism, the beta-adrenoceptor system is influenced time-dependently, whereas hypothyroidism affects the beta-adrenoceptor system independent of time.

Exocytotic stimulation promotes association of the ADP-ribosylation factor with PC12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of small molecular, monomeric GTP-binding (G) proteins, initially identified by their ability to enhance cholera toxin (CTX) ADP-ribosyltransferase activity. ARFs have been implicated in protein transport and vesicle and endosome fusion. Although several reports show that synthetic peptides of the N-terminus of ARF inhibited Ca(2+)-dependent exocytosis in permeabilized adrenal chromaffin cells, the role of ARFs in exocytosis has not been established. In this study, we investigated the translocation of ARFs to the membrane fraction from the cytosol fraction in PC12 cells after exocytotic stimulation by measuring the immunoreactivity of ARFs (with anti-ARF anti-serum and with anti-ARF3 antibodies) and enzymatic ARF activity, which enhances the CTX effect. Both the immunoreactivity and the enzymatic activity of ARF in the membrane fraction increased about twofold, significantly, after exocytotic stimulation with ATP and KCl. The translocation of ARF and noradrenaline release was observed in the presence of extracellular CaCl2, but not in the absence of CaCl2. The ARF translocated to the membrane fraction after stimulation in intact cells seemed to be an inactive, perhaps is the GDP form, because ARF did not activate CTX in the absence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As previously reported, ARF in the active, GTP gamma S-bound state bound to the membrane fractions. Thus ARF may have been active during translocation and inactivated later. The immunoreactivity of Gs alpha, one of the trimeric G proteins, was not changed before or after stimulation. These findings suggest that ARFs translocate to membranes from the cytosolic fraction after exocytotic stimulation in PC12 cells, and raise the possibility that ARFs regulate exocytosis.

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz.

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.

G-protein(S), G alpha q/G alpha 11, in the olfactory neuroepithelium of the channel catfish (Ictalurus punctatus) is altered by the herbicide, dichlobenil.

The immunohistochemical localization of G alpha q/G alpha 11 was studied in the olfactory neuroepithelium of the channel catfish. Antigenicity in the rosette was found at the apical surfaces of cells, within the apical neck regions of some cells, and within the area of the basal nerve tracts. Specific labeling was eliminated by preincubation of the G alpha q/G alpha 11 antibodies with the cognate peptide. Catfish, exposed to a 20 ppm dose of the herbicide, dichlobenil, displayed a reduction in G alpha q/G alpha 11 antigenicity. Proteins were extracted from olfactory tissues and solubilized. Using SDS-PAGE and Western blotting, bands corresponding in apparent molecular weight to a 38 kD protein were found. These data demonstrate that the herbicide may be a potent nasal olfactory toxicant in aquatic situations.