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Ribozyme approach identifies a functional association between the G protein β₁γ₇ subunits in the β-adrenergic receptor signaling pathway

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第24期274卷 17365-17371页

作者： Wang, Q Mullah, BK Robishaw, JD Penn State Univ Henry Hood MD Res Program Coll Med Weis Ctr Res Danville PA 17822 USA Appl Biosyst Inc Foster City CA 94404 USA

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effecters. However, in most cases, the specific combinations of alpha and beta gamma subunits comprising the G proteins that participate in the most common signaling pathways, such as beta-adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the beta gamma subunits that combine with the alpha subunit of G(s) are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the gamma(7) subunit of the G proteins, thereby identifying a specific role of this protein in coupling the beta-adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells, In the present study, we explored the potential utility of a ribozyme approach directed against the gamma(7) subunit to identify functional associations with a particular beta and alpha(s) subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the gamma(7) subunit, and the effects of this manipulation on levels of the beta and alpha(s) subunits were determined by immunoblot analysis. Among the five beta alpha(s) subunits detected in these cells, only the beta(1) subunit was coordinately reduced following treatment with the ribozyme directed against the gamma(7) subunit, thereby demonstrating a functional association between the beta(1) and gamma(7) subunits, The mechanism for coordinate suppression of the beta(1) subunit was due to a striking change in the half-life of the beta(1) monomer versus the beta(1) heterodimer complexed with the gamma(7) subunit, Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme dire

关键词：细胞分级分离 gtp结合蛋白质α亚单位 Gs/遗传学 gtp结合蛋白质α亚单位 Gs/代谢 gtp结合蛋白质β亚单位 gtp结合蛋白质γ亚单位 gtp结合蛋白质类/代谢半衰期异源三聚体gtp结合蛋白质类动力学沉淀素试验蛋白质结合蛋白质构象 RNA/代谢 RNA 催化/遗传学 RNA 催化/代谢 RNA 信使/代谢受体肾上腺素能β/代谢信号传导人类

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Gα₁₃ requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第20期275卷 14992-14999页

作者： Bhattacharyya, R Wedegaertner, PB Thomas Jefferson Univ Dept Microbiol & Immunol Kimmel Canc Inst Philadelphia PA 19107 USA

Most heterotrimeric G protein alpha subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of alpha(13), a G protein alpha subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant alpha(13), in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant alpha(13) retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated alpha(13) induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of alpha(13) fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of alpha(13) as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by alpha(13).

关键词：氨基酸序列氨基酸取代 COS细胞细胞系细胞膜/代谢半胱氨酸 DNA结合蛋白质类/代谢 gtp结合蛋白质α亚单位 G12-G13 鸟嘌呤核苷酸交换因子类/代谢异源三聚体gtp结合蛋白质类/化学异源三聚体gtp结合蛋白质类/代谢萤光素酶类/遗传学分子序列数据诱变定点核蛋白质类/代谢棕榈酸/代谢蛋白质加工转译后重组蛋白质类/化学重组蛋白质类/代谢序列比对血清反应因子信号传导转录因子/代谢转录遗传转染 ρgtp结合蛋白质类/代谢动物人类大鼠

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The β₃-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a G_i-dependent mechanism

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第17期274卷 12017-12022页

作者： Soeder, KJ Snedden, SK Cao, WH Della Rocca, GJ Daniel, KW Luttrell, LM Collins, S Duke Univ Med Ctr Dept Psychiat & Behav Sci Durham NC 27710 USA Duke Univ Med Ctr Dept Pharmacol Durham NC 27710 USA Duke Univ Med Ctr Dept Biochem Durham NC 27710 USA Duke Univ Med Ctr Dept Med Durham NC 27710 USA

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G;proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual G(s)/G(i) protein coupling of the beta(3)-adrenergic receptor (beta(3)AR) in 3T3-F442A adipocytes. The beta(3)AR selective agonist disodium (R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further a-fold increase in cAMP production by CL316,243, CL316,243 (5 mu M) stimulated the incorporation of 8-azido-[P-32]gtp into G alpha(s) (1.57 +/- 0.12;n = 3) and G alpha(i) (1.68 +/- 0.13;n = 4) in adipocyte plasma membranes, directly demonstrating that beta(3)AR stimulation results in G(i)-gtp exchange. The beta(3)AR-stimulated increase in 8-azido-[P-32]gtp labeling of G alpha(i) was equivalent to that obtained with the A(1)-adenosine receptor agonist N-6-cyclopentyladenosine (1.56 +/- 0.07;n = 4), whereas inclusion of unlabeled gtp (100 mu M) eliminated all binding. Stimulation of the beta(3)AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta(3)AR, demonstrating that this response required receptor coupling to Gi, Expression of the human beta(3)AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta(3)AR, ERK1/2 activation by the beta(3)AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data

关键词： 3T3细胞脂细胞/酶学肾上腺素能β激动剂/药理学钙-钙调素依赖性蛋白激酶类/代谢二恶茂类/药理学酶激活 gtp结合蛋白质α亚单位 Gi-Go/代谢脂解作用蛋白质结合受体肾上腺素能β/代谢受体肾上腺素能β3 动物人类小鼠

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The regulator of G protein signaling RGS4 selectively enhances α_2A-adreoreceptor stimulation of the gtpase activity of G_o1α and G_i2α

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第31期275卷 23693-23699页

作者： Cavalli, A Druey, KM Milligan, G Univ Glasgow Inst Biomed & Life Sci Div Biochem & Mol Biol Mol Pharmacol Grp Glasgow G12 8QQ Lanark Scotland NIAID Mol Signal Transduct Sect Lab Allerg Dis NIH Rockville MD 20852 USA

Agonist-stimulated high affinity gtpase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal gtpase activity nor agonist stimulation of the fusion proteins containing G alpha(i1) and G alpha(i3) but markedly enhanced agonist-stimulation of the proteins containing G alpha(i2) and G alpha(o1). The effect of RGS4 on the alpha(2A)-adrenoreceptor-G alpha(o1) fusion protein was concentration-dependent with EC50 of 30 +/- 3 nM and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nM RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-G alpha(o1) or alpha(2A)-adrenoreceptor-G alpha(i2) fusion proteins. Enzyme kinetic analysis of the gtpase activity of the alpha(2A)-adrenoreceptor-G alpha(o1) and alpha(2A)-adrenoreceptor-G alpha(i2) fusion proteins demonstrated that RGS4 both substantially increased gtpase V-max and significantly increased K-m of the fusion proteins for gtp. The increase in K-m for gtp was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated gtpase turnover number in the presence of 100 nM RGS4 was substantially higher for alpha(2A)-adrenoreceptor-G alpha(o1) than for alpha(2A)-adrenoreceptor-G alpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the gtpase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.

关键词：肾上腺素能α激动剂/药理学 gtp结合蛋白α亚基 Gi2 gtp结合蛋白质α亚单位 Gi-Go 异源三聚体gtp结合蛋白质类/遗传学异源三聚体gtp结合蛋白质类/代谢动力学突变原癌基因蛋白质类/遗传学原癌基因蛋白质类/代谢喹恶啉类/药理学 RGS蛋白质类/遗传学 RGS蛋白质类/代谢受体肾上腺素能α2/激动剂受体肾上腺素能α2/遗传学受体肾上腺素能α2/代谢重组融合蛋白质类/代谢猪动物

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Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第5期276卷 3010-3016页

作者： Kudlacek, O Mitterauer, T Nanoff, C Hohenegger, M Tang, WJ Freissmuth, M Kleuss, C Univ Vienna Inst Pharmacol A-1090 Vienna Austria Univ Chicago Dept Neurobiol Pharmacol & Physiol Chicago IL 60637 USA Free Univ Berlin Inst Pharmacol D-14195 Berlin Germany

The pyrophosphate (PPi) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PPi in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by G alpha (s) relieved the inhibition by foscarnet but not that by PPi, The IC50 of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines;foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase, Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.

关键词：腺苷酸环化酶/拮抗剂和抑制剂抗肿瘤联合化疗方案抗病毒药/药理学细胞培养的仓鼠亚科环AMP/代谢环GMP/代谢环磷酰胺二聚作用二磷酸盐类/化学二磷酸盐类/代谢二磷酸盐类/药理学多柔比星酶抑制剂/药理学膦甲酸/药理学 gtp结合蛋白质α亚单位 Gs/代谢鸟苷酸环化酶/拮抗剂和抑制剂同工酶类/拮抗剂和抑制剂膜蛋白质类/代谢长春新碱动物人类

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Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第1期275卷 288-296页

作者： An, SZ Zheng, YH Bleu, T Univ Calif San Francisco Dept Med San Francisco CA 94143 USA

Sphingosine l-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Contradictory reports propose that S1P acts as either an intracellular second messenger or an extracellular ligand for cell-surface receptors. Hence, the precise signaling mechanisms mediating the diverse cellular effects of S1P remain to be determined. Here, we investigate whether S1P stimulation of cell. proliferation, survival, and related signaling events can be mediated by the recently cloned Edg family members of G protein-coupled receptors. We observed that S1P treatment significantly increased proliferation of HTC4 hepatoma cells stably transfected with human S1P receptor Edg3 or Edg5, which was attributable to stimulation of cell growth and inhibition of apoptosis caused by serum starvation. Edg3 and Edg5 transduced S1P-evoked signaling events relevant to cell proliferation and survival, including activation of the ERK/MAP kinases, and immediate-early induction of c-Jun and c-Fos. Transcriptional activation of reporter genes for the c-fos promoter and the serum response element by Edg3 and Edg5 transfected in Jurkat cells was inhibited by pertussis toxin and C3 exoenzyme, implicating G(i/o)- and Rho-dependent pathways. Our data also indicated that Edg3 and Edg5 mediated the serum response element activation through transcriptional factors Elk-1 and serum response factor. Thus, specific G protein-coupled receptors Edg3 and Edg5 account for, at least in part, S1P-induced cell proliferation, survival, and related signaling events.

关键词：细胞分裂细胞存活克隆分子 DNA结合蛋白质类/代谢 gtp结合蛋白质α亚单位 Gi-Go gtp结合蛋白质类/代谢 I-κB蛋白质类溶血磷脂素类丝裂原活化蛋白激酶1/代谢丝裂原活化蛋白激酶3 丝裂原激活蛋白激酶类/代谢分子序列数据核蛋白质类/代谢原癌基因蛋白质类/代谢原癌基因蛋白质c-fos/代谢原癌基因蛋白质c-jun/代谢受体细胞表面/遗传学受体细胞表面/代谢受体 G-蛋白偶联受体溶血磷脂血清反应因子信号传导鞘氨醇/类似物和衍生物鞘氨醇/代谢转录因子AP-1/代谢转录因子 ets结构域蛋白Elk1 ρgtp结合蛋白质类/代谢人类

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Adenylyl cyclase, a coincidence detector for nitric oxide

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第27期274卷 18887-18892页

作者： McVey, M Hill, J Howlett, A Klein, C St Louis Univ Sch Med Dept Biochem & Mol Biol St Louis MO 63104 USA St Louis Univ Sch Med Dept Pharmacol & Physiol Sci St Louis MO 63104 USA

Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V-max of the enzyme without altering the K-m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.

关键词：前列地尔/药理学印迹法 RNA 细胞膜/酶学剂量效应关系药物酶抑制剂/药理学 gtp结合蛋白质α亚单位 Gi-Go/代谢动力学一氧化氮/代谢一氧化氮供体/药理学青霉胺/类似物和衍生物青霉胺/药理学 S-亚硝基-N-乙酰基青霉胺肿瘤细胞培养的动物

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Regulation of G_αi palmitoylation by activation of the 5-hydroxytryptamine-1A receptor

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第31期275卷 23516-23522页

作者： Chen, CA Manning, DR Univ Penn Sch Med Dept Pharmacol Philadelphia PA 19104 USA

Nearly all alpha subunits of heterotrimeric gtp-binding regulatory proteins (G proteins) are palmitoylated at cysteine residues near the N terminus. A regulated cycle of palmitoylation could provide a mechanism for modulating G protein signaling by affecting protein interactions and localization of the subunit, In the present studies we utilized both [H-3]palmitate incorporation and pulse chase techniques to address the dynamics of alpha(i) palmitoylation in Chinese hamster ovary cells. Both techniques demonstrated a dose- and time-dependent change in [H-3]palmitate labeling of alpha(i) upon activation of stably expressed 5-hydroxytryptamine-1A receptors by the agonist (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (DPAT), with an EC50 of similar to 10 nM, For the incorporation assay, DPAT elicited an approximate doubling in labeling at the earliest time point measured. For the pulse-chase assay, DPAT promoted a significant loss of radiolabel almost equally as fast. These data demonstrate that the exchange of palmitate on cui is increased upon stimulation of 5-hydroxytryptamine-1A receptors through the combined processes of depalmitoylation and palmitoylation, These results provide the basis for extending the concept of regulated exchange of palmitate beyond G(s) and provide a framework for exploring the specific functional attributes of the palmitoylated and depalmitoylated forms of subunit.

关键词： 8-羟-2-(二-n-丙胺)四氢萘/药理学氨基吡啶类/药理学 CHO细胞仓鼠亚科 gtp结合蛋白质α亚单位 Gi-Go/代谢 gtp结合蛋白质α亚单位 Gs/代谢棕榈酸/代谢哌嗪类/药理学蛋白质加工转译后受体血清素/代谢受体血清素 5-HT1 信号传导毒力因子鲍特菌属/药理学动物人类

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Interaction with Gβγ is required for membrane targeting and palmitoylation of Gα_s, and Gα_q

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第2期275卷 1327-1336页

作者： Evanko, DS Thiyagarajan, MM Wedegaertner, PB Thomas Jefferson Univ Dept Microbiol & Immunol Philadelphia PA 19107 USA Thomas Jefferson Univ Kimmel Canc Inst Philadelphia PA 19107 USA

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits, For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein beta gamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to beta gamma were mutated to create a series of beta gamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy, The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation, These findings identify binding to beta gamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of beta gamma binding-deficient alpha(s) subunits.

关键词：氨基酸序列细胞系细胞膜/代谢保守序列环AMP/代谢二聚作用 gtp结合蛋白质α亚单位 Gs/化学 gtp结合蛋白质α亚单位 Gs/遗传学 gtp结合蛋白质α亚单位 Gs/代谢 gtp结合蛋白质类/化学 gtp结合蛋白质类/遗传学 gtp结合蛋白质类/代谢肌醇磷酸类/代谢大分子物质分子序列数据诱变定点棕榈酸/代谢蛋白质加工转译后重组蛋白质类/化学重组蛋白质类/代谢序列比对序列同源性氨基酸转染人类

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Adenosine A_2A receptors are colocalized with and activate G_olf in rat striatum

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MOLECULAR PHARMACOLOGY 2000年第4期58卷 771-777页

作者： Kull, B Svenningsson, P Fredholm, BB Karolinska Inst Dept Physiol & Pharmacol Sect Mol Neuropharmacol S-17177 Stockholm Sweden

In situ hybridization with cRNA probes showed A(2A) receptor and G(olf) mRNAs to be abundantly expressed in caudate putamen, nucleus accumbens, and olfactory tubercle, whereas G(s) mRNA shows a comparatively low expression in regions expressing A(2A) receptors. In caudate putamen, 49% of the medium-sized neuron-like cells exhibited a strong signal for adenosine A(2A) receptor mRNA, and 98% showed a strong signal for G(olf) mRNA. In contrast, G(s) mRNA was found in only 12% of the medium-sized neuron-like cells in caudate putamen. The coexpression of adenosine A(2A) receptor mRNA with that of G(olf) or G(s) mRNAs was studied with double in situ hybridization. A large majority (91-95%) of the neurons in caudate-putamen that contained adenosine A(2A) receptor mRNA also expressed G(olf) mRNA, whereas only 3 to 5% of the neurons with adenosine A(2A) receptor mRNA coexpressed G(s) mRNA. The A(2A) receptor agonist CGS 21680 [2-[p-(2-carbonylethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine] dose dependently activated Golf subunits in striatal membranes as shown by photolabeling with [ alpha-P-32]m-acetylanilido-gtp followed by immunoprecipitation with a specific antibody against G(olf). Transfection of G(olf) cDNA into Chinese hamster ovary cells, which stably express human adenosine A(2A) receptors, led to an increased efficacy of CGS 21680, as evidenced by a stronger cAMP response, indicating that activation of G(olf) by A(2A) receptors leads to a biological signal. In conclusion, these results provide anatomical and biochemical evidence that adenosine A(2A) receptors stimulate G(olf) rather than G(s) in striatum.

关键词：细胞膜/代谢纹状体/代谢环AMP/代谢 gtp结合蛋白质α亚单位 gtp结合蛋白质α亚单位 Gs/遗传学 gtp结合蛋白质α亚单位 Gs/代谢异源三聚体gtp结合蛋白质类/遗传学异源三聚体gtp结合蛋白质类/代谢前脑/代谢 RNA 信使/代谢大鼠 Sprague-Dawley 受体腺苷A2A 受体嘌呤能P1/遗传学受体嘌呤能P1/代谢动物男(雄)性大鼠

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