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1.
G protein hyperactivation of the Caenorhabditis elegans adenylyl cyclase SGS-1 induces neuronal degeneration. 总被引:1,自引:0,他引:1
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Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability. 相似文献
2.
Klemke M Pasolli HA Kehlenbach RH Offermanns S Schultz G Huttner WB 《The Journal of biological chemistry》2000,275(43):33633-33640
In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase. 相似文献
3.
Toshiaki Ebina Jun-ichi Kawabe Toshiaki Katada Shigeo Ohno Charles J. Homcy Yoshihiro Ishikawa 《Journal of cellular biochemistry》1997,64(3):492-498
Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc. 相似文献
4.
In the present study, we investigated the involvement of betagamma subunits of G(q/11) in the muscarinic M(1) receptor-induced potentiation of corticotropin-releasing hormone (CRH)-stimulated adenylyl cyclase activity in membranes of rat frontal cortex. Tissue exposure to either one of two betagamma scavengers, the QEHA fragment type II adenylyl cyclase and the GDP-bound form of the alpha subunit of transducin, inhibited the muscarinic M(1) facilitatory effect. Moreover, like acetylcholine (ACh), exogenously added betagamma subunits of transducin potentiated the CRH-stimulated adenylyl cyclase activity, and this effect was not additive with that elicited by ACh. Western blot analysis indicated the expression in frontal cortex of both type II and type IV adenylyl cyclases, two isoforms stimulated by betagamma subunits in synergism with activated G(s). The M(1) receptor-induced enhancement of the adenylyl cyclase response to CRH was counteracted by the G(q/11) antagonist GpAnt-2A but not by GpAnt-2, a preferential G(i/o) antagonist. In addition, the muscarinic facilitatory effect was inhibited by membrane preincubation with antiserum directed against the C terminus of the alpha subunit of G(q/11), whereas the same treatment with antiserum against either G(i1/2) or G(o) was without effect. These data indicate that in membranes of rat frontal cortex, activation of muscarinic M(1) receptors potentiates CRH-stimulated adenylyl cyclase activity through betagamma subunits of G(q/11). 相似文献
5.
Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. 总被引:15,自引:0,他引:15
We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively. 相似文献
6.
Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity. 相似文献
7.
Dopamine receptor signaling 总被引:13,自引:0,他引:13
Neve KA Seamans JK Trantham-Davidson H 《Journal of receptor and signal transduction research》2004,24(3):165-205
8.
Iwamoto T Okumura S Iwatsubo K Kawabe J Ohtsu K Sakai I Hashimoto Y Izumitani A Sango K Ajiki K Toya Y Umemura S Goshima Y Arai N Vatner SF Ishikawa Y 《The Journal of biological chemistry》2003,278(19):16936-16940
Various neurotransmitters, such as dopamine, stimulate adenylyl cyclase to produce cAMP, which regulates neuronal functions. Genetic disruption of the type 5 adenylyl cyclase isoform led to a major loss of adenylyl cyclase activity in a striatum-specific manner with a small increase in the expression of a few other adenylyl cyclase isoforms. D1 dopaminergic agonist-stimulated adenylyl cyclase activity was attenuated, and this was accompanied by a decrease in the expression of the D1 dopaminergic receptor and G(s)alpha. D2 dopaminergic agonist-mediated inhibition of adenylyl cyclase activity was also blunted. Type 5 adenylyl cyclase-null mice exhibited Parkinsonian-like motor dysfunction, i.e. abnormal coordination and bradykinesia detected by Rotarod and pole test, respectively, and to a lesser extent locomotor impairment was detected by open field tests. Selective D1 or D2 dopaminergic stimulation improved some of these disorders in this mouse model, suggesting the partial compensation of each dopaminergic receptor signal through the stimulation of remnant adenylyl cyclase isoforms. These findings extend our knowledge of the role of an effector enzyme isoform in regulating receptor signaling and neuronal functions and imply that this isoform provides a site of convergence of both D1 and D2 dopaminergic signals and balances various motor functions. 相似文献
9.
10.
R H Paulssen E J Paulssen K M Gautvik J O Gordeladze 《European journal of biochemistry》1992,204(1):413-418
The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C. 相似文献
11.
Tang T Gao MH Roth DM Guo T Hammond HK 《American journal of physiology. Heart and circulatory physiology》2004,287(5):H1906-H1912
Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart. 相似文献
12.
The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol. 相似文献
13.
To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor. 相似文献
14.
Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein. 相似文献
15.
The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum. 相似文献
16.
17.
Chunfa Huang John R. Hepler Linda T. Chen Alfred G. Gilman Richard G.W. Anderson Susanne M. Mumby 《Molecular biology of the cell》1997,8(12):2365-2378
There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction. 相似文献
18.
The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system. 相似文献
19.
Yang J Wu J Jiang H Mortensen R Austin S Manning DR Woulfe D Brass LF 《The Journal of biological chemistry》2002,277(48):46035-46042
Platelet responses at sites of vascular injury are regulated by intracellular cAMP levels, which rise rapidly when prostacyclin (PGI(2)) is released from endothelial cells. Platelet agonists such as ADP and epinephrine suppress PGI(2)-stimulated cAMP formation by activating receptors coupled to G(i) family members, four of which are present in platelets. To address questions about the specificity of receptor:G protein coupling, the regulation of cAMP formation in vivo and the contribution of G(i)-mediated pathways that do not involve adenylyl cyclase, we studied platelets from mice that lacked the alpha subunits of one or more of the three most abundantly expressed G(i) family members and compared the results with platelets from mice that lacked the PGI(2) receptor, IP. As reported previously, loss of G(i2)alpha or G(z)alpha inhibited aggregation in response to ADP and epinephrine, respectively, producing defects that could not be reversed by adding an adenylyl cyclase inhibitor. Platelets that lacked both G(i2)alpha and G(z)alpha showed impaired responses to both agonists, but the impairment was no greater than in the individual knockouts. Loss of G(i3)alpha had no effect either alone or in combination with G(z)alpha. Loss of either G(z)alpha or G(i2)alpha impaired the ability of ADP and epinephrine to inhibit PGI(2)-stimulated adenylyl cyclase activity and caused a 40%-50% rise in basal cAMP levels, whereas loss of G(i3)alpha did not. Conversely, deletion of IP abolished responses to PGI(2) and caused cAMP levels to fall by 30%, effects that did not translate into enhanced responsiveness to agonists ex vivo. From these results we conclude that 1) cAMP levels in circulating platelets reflect ongoing signaling through G(i2), G(z), and IP, but not G(i3); 2) platelet epinephrine (alpha(2A)-adrenergic) and ADP (P2Y12) receptors display strong preferences among G(i) family members with little evidence of redundancy; and 3) these receptor preferences do not extend to G(i3). Finally, the failure of ADP and epinephrine to inhibit basal, as opposed to PGI(2)-stimulated, cAMP formation highlights the need during platelet activation for G(i) signaling pathways that involve effectors other than adenylyl cyclase. 相似文献
20.
Haney SA Xu J Lee SY Ma CL Duzic E Broach JR Manfredi JP 《Molecular genetics and genomics : MGG》2001,265(6):1120-1128
We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs. 相似文献