A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins.

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.

Zn2+-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin.

Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Antigen targeting to CD11b+ dendritic cells in association with TLR4/TRIF signaling promotes strong CD8+ T cell responses.

Deciphering the mechanisms that allow the induction of strong immune responses is crucial to developing efficient vaccines against infectious diseases and cancer. Based on the discovery that the adenylate cyclase from Bordetella pertussis binds to the CD11b/CD18 integrin, we developed a highly efficient detoxified adenylate cyclase-based vector (CyaA) capable of delivering a large variety of Ags to the APC. This vector allows the induction of protective and therapeutic immunity against viral and tumoral challenges as well as against transplanted tumors in the absence of any added adjuvant. Two therapeutic vaccine candidates against human papilloma viruses and melanoma have been developed recently, based on the CyaA vector, and are currently in clinical trials. We took advantage of one of these highly purified vaccines, produced under good manufacturing practice-like conditions, to decipher the mechanisms by which CyaA induces immune responses. In this study, we demonstrate that CyaA binds both human and mouse CD11b(+) dendritic cells (DCs) and induces their maturation, as shown by the upregulation of costimulatory and MHC molecules and the production of proinflammatory cytokines. Importantly, we show that DCs sense CyaA through the TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß pathway, independent of the presence of LPS. These findings show that CyaA possesses the intrinsic ability to not only target DCs but also to activate them, leading to the induction of strong immune responses. Overall, this study demonstrates that Ag delivery to CD11b(+) DCs in association with TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß activation is an efficient strategy to promote strong specific CD8(+) T cell responses.

Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis.

HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.

A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits.

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.

Augmentation of receptor-mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a Gbetagamma subunit-independent manner.

We previously demonstrated that the mouse EP3beta receptor and its C-terminal tail-truncated receptor (abbreviated T-335) expressed in Chinese hamster ovary cells showed agonist-dependent and fully constitutive Gi activity in forskolin-stimulated cAMP accumulation, respectively. Here we examined the effect of the EP3beta receptor or T-335 receptor on adenylyl cyclase activity stimulated by the Gs-coupled EP2 subtype receptor in COS-7 cells. As a result, sulprostone, a selective EP3 agonist, dose dependently augmented butaprost-stimulated adenylyl cyclase activity in EP3beta receptor- or T-335 receptor-expressing COS-7 cells. However, such adenylyl cyclase augmentation was not attenuated by either pertussis toxin treatment or expression of the PH domain of rat betaARK1, which serves as a scavenger of Gbetagamma subunits, but was partially attenuated by treatment with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester, an intracellular Ca(2+) chelator, or W-7, a calmodulin inhibitor. These findings suggest that the C-terminal tail of the EP3beta receptor is not essentially involved in activation of EP2 receptor-stimulated adenylyl cyclase in a Ca(2+)/calmodulin-dependent but Gbetagamma subunit-independent manner.

Autoreceptor preference of dopamine D2 receptor agonists correlates with preferential coupling to cyclic AMP.

Dopamine autoreceptors control the synaptic release and turnover of dopamine. Some dopamine agonists display a preference for modulation of autoreceptor functions rather than postsynaptic-driven behaviors. However, the nature of this apparent selectivity is still elusive. To investigate this property, we have used an heterologous expression system in which D2S receptors are coupled to both inhibition of cyclic AMP levels and stimulation of inositol triphosphate production. We show that D2-like receptor agonists display distinct potencies on these two second messenger pathways. Moreover, a strong correlation is observed between the potency of agonists to interact with adenylate cyclase and their potency to modulate autoreceptor functions. Such a correlation does not show up with the phospholipase C pathway. This suggests that autoreceptor preference of D2-like receptor agonists may be driven by a preferential interaction with a second messenger system.

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons.

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.

Altered adenylyl cyclase responsiveness subsequent to point mutations of Asp 128 in the third transmembrane domain of the delta-opioid receptor.

delta-Opioid receptors belong to the superfamily of G protein-coupled receptors, characterized by seven putative transmembrane domains, and have been shown to interact with a host of effector systems. It has been suggested that the charge on the conserved aspartic acid residue at position 128 in transmembrane domain 3 of the delta-opioid receptor contributes to both the conformation of the receptor binding pocket and the molecular rearrangements which accompany the establishment of high-affinity states of the receptor. In light of this, we used site-directed mutagenesis to determine whether this residue participates in the transmission of signals to adenylyl cyclase, the effector with which opioid receptors have been classically associated. Substitution of this aspartic acid (D128) for the neutral amino acid alanine, or the protonated amino acids lysine and histidine, constitutively couples the receptor to adenylyl cyclase, as evidenced by a curtailed response to forskolin stimulation in transfected cells. In addition, this constitutive activity can be blocked by pretreatment of the transfected cells with pertussis toxin. Interestingly, naloxone blocks this effect in cells expressing the D128A mutant, but acts as an agonist at the D128K mutant. Our findings support the hypothesis that the interaction between agonist and receptor promotes conformational changes that may be mimicked, at least in part, by mutation of the aspartate residue at position 128. Furthermore, these changes appear to be involved not only in receptor activation, but also in the functional discrimination between agonists and antagonists.

Effects of UTP on Na+, Cl- and K+ transport in primary cultures from human sweat gland coils.

Extracellular ATP and UTP can increase membrane permeability in the sweat gland, but the intracellular signalling regulating the response to these agonists is poorly understood. Stimulation of Cl- transport by nucleotides has been suggested as a pharmacological therapy to improve Cl- secretion in patients with cystic fibrosis. In the present study, regulation of Na+, Cl- and K+ transport in primary cultures of cells from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. Stimulation with 200 microM UTP for 2 min at room temperature caused a significant increase in intracellular Na but did not affect Cl and K. After 5 min, the Na concentration was still increased, but now also a significant decrease in Cl and K was observed, indicating an increase in Cl- and K+ permeability. The effect of UTP on Cl- secretion was enhanced in Mg2+-deficient buffer, indicating that the response is elicited by the extracellular fully ionized form of UTP (UTP4+), but not by MgUTP2+. The effects of UTP were abolished in Ca2+-deficient buffer supplemented with EGTA. Alloxan, an adenylate cyclase inhibitor, did not inhibit the response to UTP. These results indicate that the membrane Cl- and K+ permeability elicited by UTP in primary coil cell cultures is Ca2+-dependent. The response to UTP did not attenuate at 8 degrees C, suggesting that it could be activated, in part, via ligand-gated ion channels. The effect of UTP was not decreased in the presence of ouabain. Pre-treatment of the cells with pertussis toxin (24 h) had minor effects on Cl- secretion activated by UTP, indicating a role for G proteins in the UTP activation of Cl- secretion.

Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway.

Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257-264 CD8+ epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD8+ T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca2+ and independent of vacuolar acidification. Moreover, inhibitors of the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation pathway is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconventional delivery of an exogenous Ag into the conventional cytosolic pathway.

Adenosine A1 receptor-dependent and -independent effects of the allosteric enhancer PD 81,723.

The 2-amino-3-benzoylthiophene PD 81,723 has been shown to exhibit allosteric enhancement of adenosine A1 receptor binding and function. The aim of this study was to clarify the mechanism of this effect using membranes purified from rat brain and Chinese hamster ovary (CHO)-A1 cells that stably express the rat adenosine A1 receptor as well as intact CHO-A1 and nontransfected CHO cells. In membranes containing 100 microM magnesium, (2-amino-4, 5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81, 723) significantly increased the affinity of the adenosine A1 receptor agonist, cyclopentyladenosine, for the low-affinity receptor without affecting high-affinity binding or Bmax. In intact cells, PD 81,723 inhibited basal adenylyl cyclase (AC) activity as well as forskolin-, cholera toxin-, and pertussis toxin-stimulated AC activity in CHO-A1 and CHO cells. Basal AC activity was inhibited 49% in CHO and 82% in CHO-A1 cells by 30 microM PD 81,723. In CHO-A1 cells, half-maximal inhibition of forskolin-stimulated AC occurred at 5 microM PD 81,723 compared to 10 microM in CHO cells. Cholera toxin-stimulated AC was reduced 90% in both CHO and CHO-A1 cells by 30 microM PD 81,723. At the same concentration of PD 81,723, pertussis toxin-stimulated AC activity was reduced 86% (CHO-A1) and 77% (CHO). [3H]forskolin was displaced from purified rat liver AC by PD 81,723 with an IC50 of 96 microM. These results demonstrate that two mechanisms appear to contribute to the observed effects of PD 81, 723. One mechanism is allosteric enhancement of adenosine A1 receptor function. Results from transfected and nontransfected cells suggest that PD 81,723 also inhibits AC directly by binding to the catalytic unit at or near the forskolin-binding site.

Acetylcholine-induced biphasic effect on the maximum upstroke velocity of action potential in mouse right atria: interaction with beta-adrenergic signaling cascade.

Several lines of evidence suggest the molecular and functional entity of muscarinic M1 receptors in mammalian heart. We have reported that acetylcholine (ACh) reduces the maximum upstroke velocity of action potential (Vmax) through activation of muscarinic M1 receptors, which is followed by a muscarinic M2 receptor-mediated increase. The present study sought to determine whether activation of beta-adrenergic receptors modulates the muscarinic M1 and M2 receptor-mediated effects on Vmax in isolated mouse right atria. Intracellular recordings of spontaneous action potential were done using the conventional glass microelectrode technique. Isoproterenol (3 nM) completely antagonized ACh (5 microM)-induced reduction in Vmax. The antagonism was accompanied by a subsequent increase in Vmax. Propranolol (0.3 microM) abolished the effects of isoproterenol on ACh-induced changes in Vmax. Isoproterenol antagonized McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride) (300 microM, a muscarinic M1 receptor agonist)-induced reduction in Vmax. Oxotremorine (0.03 microM), a muscarinic M2 receptor agonist, did not affect Vmax by itself, but significantly increased it in the presence of 3 nM isoproterenol. The effects of isoproterenol were mimicked by cholera toxin (100 nM, 1 hr), a Gs-protein activator, and forskolin (10 nM), a direct activator of adenylyl cyclase. H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide++ +, 1 microM), a selective protein kinase (PK)-A inhibitor, abolished the antagonism by isoproterenol of ACh-induced reduction in Vmax. The present results suggest that activation of the beta-adrenergic-Gs-adenylyl cyclase system antagonizes ACh-induced reduction (muscarinic M1-mediated) and potentiates the subsequent increase (muscarinic M2 receptor-mediated) in Vmax. The beta-adrenergic antagonism of ACh-induced reduction in Vmax may involve cross-talk between PK-A and PK-C signaling pathways.

Identification and characterization of muscarinic receptors potentiating the stimulation of adenylyl cyclase activity by corticotropin-releasing hormone in membranes of rat frontal cortex.

In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-sila-difenidol, whereas the M2 and M4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [35S]GTPgammaS binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins.

Chronic treatment with dexmedetomidine desensitizes alpha 2-adrenergic signal transduction.

Tolerance to the hypnotic response was induced in rats by chronically infusing dexmedetomidine, a novel alpha 2-adrenergic agonist. The alpha 2-adrenoceptor affinity for dexmedtomidine and para-iodoclonidine was significantly reduced in tolerant rats, while Bmax was uncharged. The ability of pertussis toxin (PTX) to ribosylate guanine nucleotide regulatory proteins (G proteins) ex vivo was reduced in tolerant rats; the quantity of PTX-sensitive G proteins was unchanged. Forskolin-stimulated adenylyl cyclase was less sensitive to inhibition by dexmedetomidine in the tolerant rats; however, acute intraperitoneal injection of dexmedetomidine still reduced cyclic adenosine monophosphate levels in tolerant rats. Both the decrease in ribosylation and the lower alpha 2-adrenoceptor binding affinity may reflect a decrease in the ability of the G protein to couple to the alpha 2-adrenoceptors in the locus coeruleus of tolerant rats. In this state, the alpha 2 adrenoceptors are less capable of transducing the effector response (inhibition of adenylyl cyclase).

Receptor-induced beta gamma release from fatty acylation-deficient mutants of G alpha z.

The neuronal-specific G protein Gz is known to interact with a large variety of receptors for neurotransmitters and hormones. Fatty acylations on the N-terminus of the alpha subunit of Gz (alpha z) provide anchorage to the plasma membrane. Fatty acylation-deficient mutants of alpha z have previously been shown to exhibit altered signaling properties. Since the N-terminus of alpha z is likely to play a critical role in beta gamma binding, we examined the ability of these mutants to interact with beta gamma subunits by means of receptor-mediated stimulation of beta gamma-sensitive type II adenylyl cyclase. Our results indicate that lack of myristoylation, but not lack of palmitoylation, impaired the ability of alpha z to mediate receptor-induced release of beta gamma subunits.

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C.

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.

Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release.

The GT1-7 cell line of immortalized GnRH neurons has been shown to express receptors for GnRH, LH, and prolactin, as well as a variety of other hormones and transmitters. Treatment of GT1-7 cells with hCG caused a dose-dependent increase in cAMP production, with a rapid increase during the first 15 min and a subsequent decrease that was prevented by pre-treatment with pertussis toxin. Furthermore, the stimulatory effect of cholera toxin on cAMP production was inhibited by hCG in a dose-dependent manner. These data indicate that the LH receptors expressed in GT1-7 cells are coupled to adenylyl cyclase both stimulatory (Gs) and inhibitory (Gi) proteins. In perifused cell cultures, treatment with forskolin and 8-bromo cAMP increased the amplitude of spontaneous GnRH release. However, treatment with nanomolar concentrations of hCG abolished pulsatile GnRH release from both GT1-7 cells and rat hypothalamic cells. The similarity of hCG action on pulsatile GnRH release to that of extracellular Ca2+ depletion and calcium channel antagonists, and its partial resistance to potassium-induced depolarization, suggest that it results from inhibition of plasma-membrane ion channel activity. It is probable that the inhibitory action of hCG on pulsatile GnRH release is responsible for initiation of the suppression of pituitary LH secretion during pregnancy.

Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists.

Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.