RESUMO
To investigate whether a particular receptor subtype can be coupled to multiple effector systems, recombinant M2 muscarinic receptors were expressed in cells lacking endogenous receptor. The muscarinic agonist carbachol both inhibited adenylyl cyclase and stimulated phosphoinositide hydrolysis. The stimulation of phosphoinositide hydrolysis was significantly less efficient and more dependent on receptor levels than the inhibition of adenylyl cyclase. Both responses were mediated by guanine nucleotide binding proteins, as evidenced by their inhibition by pertussis toxin; the more efficiently coupled adenylyl cyclase response was significantly more sensitive. Thus, individual subtypes of a given receptor are capable of regulating multiple effector pathways.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/metabolismo , Toxina Adenilato Ciclase , Animais , Carbacol/farmacologia , Linhagem Celular , Cricetinae , AMP Cíclico/biossíntese , Regulação da Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Oxotremorina/farmacologia , Toxina Pertussis , Proteínas Recombinantes , Tionucleotídeos/metabolismo , Fatores de Virulência de Bordetella/metabolismoRESUMO
A partial amino acid sequence obtained for porcine atrial muscarinic acetylcholine receptor was used to isolate complementary DNA clones containing the complete receptor coding region. The deduced 466-amino acid polypeptide exhibits extensive structural and sequence homology with other receptors coupled to guanine nucleotide binding (G) proteins (for example, the beta-adrenergic receptor and rhodopsins); this similarity predicts a structure of seven membrane-spanning regions distinguished by the disposition of a large cytoplasmic domain. Stable transfection of the Chinese hamster ovary cell line with the atrial receptor complementary DNA leads to the binding of muscarinic antagonists in these cells with affinities characteristic of the M2 receptor subtype. The atrial muscarinic receptor is encoded by a unique gene consisting of a single coding exon and multiple, alternatively spliced 5' noncoding regions. The atrial receptor is distinct from the cerebral muscarinic receptor gene product, sharing only 38% overall amino acid homology and possessing a completely nonhomologous large cytoplasmic domain, suggesting a role for the latter region in differential effector coupling.
Assuntos
Receptores Muscarínicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Éxons , Proteínas de Ligação ao GTP/metabolismo , Átrios do Coração/análise , Técnicas de Imunoadsorção , Proteínas de Membrana , Peso Molecular , Hibridização de Ácido Nucleico , Fragmentos de Peptídeos/metabolismo , Quinuclidinil Benzilato/metabolismo , Receptores Muscarínicos/metabolismo , Homologia de Sequência do Ácido Nucleico , Suínos , TransfecçãoRESUMO
BACKGROUND: One of the principal mechanisms by which G-protein-coupled receptors evoke cellular responses is through the activation of phospholipase C (PLC) and the subsequent release of Ca2+ from intracellular stores. Receptors that couple to pertussis toxin (PTX)-insensitive G proteins typically evoke large increases in PLC activity and intracellular Ca2+ release. In contrast, receptors that use only PTX-sensitive G proteins usually generate weak PLC-dependent responses, but efficiently regulate a second effector enzyme, adenylyl cyclase. For example, in many cell types, agonist binding by the m4 muscarinic acetylcholine receptor (m4 receptor) results in a strong inhibition of adenylyl cyclase and very little stimulation of PLC activity or release of intracellular Ca2+. We have investigated whether the weak, PTX-sensitive stimulation of PLC activity by the m4 receptor can play a significant role in the generation of cellular responses. RESULTS: We report here that PTX-sensitive Ca2+ release mediated by the m4 receptor in transfected Chinese hamster ovary cells is greatly enhanced when endogenous purinergic receptors simultaneously activate a PTX-insensitive signaling pathway. Furthermore, m4-receptor-induced transcription of the c-fos gene (a Ca(2+)-sensitive response) is similarly potentiated when purinergic receptors are coactivated. These enhanced m4-receptor-dependent Ca2+ responses do not require an influx of external Ca2+, and occur in the absence of detectable purinergic-receptor-stimulated Ca2+ release; they apparently require the activation of both PTX-sensitive and PTX-insensitive G-protein pathways. Measurements of phosphoinositide hydrolysis indicate that the enhancement of m4-receptor-mediated Ca2+ signaling by purinergic receptors is due to a synergistic increase in agonist-stimulated PLC activity. CONCLUSIONS: These studies demonstrate that the potency of m4-receptor-mediated PLC signaling is highly dependent upon the presence or absence of other PLC-activating agonists. The ability of the m4 receptor to evoke a strong, but conditional, activation of PLC may allow this type of receptor to participate in a coincidence-detection system that amplifies simultaneous PLC-activating signals through a mechanism involving crosstalk between PTX-sensitive and PTX-insensitive G-protein pathways.
Assuntos
Receptores Muscarínicos/metabolismo , Fosfolipases Tipo C/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Genes fos , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Ewing family tumors result from the effects of chromosomal translocations that fuse the Ewing sarcoma (EWS) gene to various genes encoding transcription factors. The resulting chimeric EWS fusion proteins are transcriptional activators with transforming potential that have received much study. By contrast, the cellular function of somatic EWS remains obscure. EWS belongs to a family of RNA-binding proteins thought to play role in RNA synthesis or processing. Here, we show that EWS interacts with Pyk2, a protein tyrosine kinase implicated in a variety of signal transduction processes. G-protein-coupled receptor signaling and other stimuli of Pyk2 kinase activity significantly block the interaction between EWS and Pyk2. Furthermore, as assessed by sucrose gradient centrifugation, EWS partitions with dense ribosome-containing fractions in a manner that is enhanced by signaling from the G-protein-coupled m1 muscarinic acetylcholine receptor (mAChR). We conclude that extranuclear EWS is a previously unrecognized target of G-protein-coupled receptor regulation.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Muscarínicos/metabolismo , Ribonucleoproteínas/metabolismo , Sarcoma de Ewing , Animais , Quinase 2 de Adesão Focal , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Células PC12 , Proteína EWS de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.
Assuntos
Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Humanos , Fatores de Transcrição NFATC , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genéticaRESUMO
The regulation of cellular signal transduction and growth by four human muscarinic acetylcholine receptor (mAChR) subtypes has been studied comparatively. The four mAChRs fall into two functional sub-groups, based on their primary effects on second messenger formation; two of the receptors strongly inhibit adenylyl cyclase activity, whereas the other two strongly stimulate PI hydrolysis. Studies on mAChR regulation of two cellular events involved in cellular growth regulation, the transcription of proto-oncogene c-fos and DNA synthesis, indicate that these events are efficiently activated by those mAChRs which couple primarily to phospholipase C.
Assuntos
Divisão Celular , Receptores Muscarínicos/fisiologia , Transdução de Sinais , Humanos , Proto-Oncogene MasRESUMO
Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.
Assuntos
Proteínas de Ligação ao GTP/farmacologia , Frequência Cardíaca/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Receptores Muscarínicos/fisiologia , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Ativação do Canal Iônico/fisiologia , Oócitos/fisiologia , Canais de Potássio/farmacologia , XenopusRESUMO
Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Given their critical role in controlling cell membrane potential, potassium channels are frequently the targets of modulatory signals from many different G protein-coupled receptors. However, due to the heterogeneity of potassium channel expression in vivo, it has been difficult to determine the molecular mechanisms governing the regulation of molecularly defined potassium channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the m1 muscarinic acetylcholine receptor (mAChR) potently suppresses a cloned delayed rectifier potassium channel, termed RAK, through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the RAK protein. In contrast, we found that RAK channel activity is strongly enhanced following agonist activation of beta2-adrenergic receptors; this effect requires a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. These results demonstrate that a specific type of potassium channel that is widely expressed in the mammalian brain and heart is subject to both positive and negative regulation by G protein-dependent pathways.
Assuntos
Canais de Potássio/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Adrenérgicos beta 2/fisiologia , Receptores Muscarínicos/fisiologia , Sequência de Aminoácidos , Animais , Receptores ErbB/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Receptor Muscarínico M1 , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , XenopusRESUMO
The G protein-coupled inwardly rectifying K+ channel, GIRK1/GIRK4, can be activated by receptors coupled to the Galpha(i) subunit. An opposing role for Galpha(q) receptor signaling in GIRK regulation has only recently begun to be established. We have studied the effects of m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is known to mobilize calcium and activate protein kinase C (PKC) by a Galpha(q)-dependent mechanism, on whole cell GIRK1/4 currents in Xenopus oocytes. We found that stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of Gbetagamma subunits attenuates this effect, suggesting that increased binding of Gbetagamma to the GIRK channel can effectively compete with the G(q)-mediated inhibitory signal. This G(q) signal requires the use of second messenger molecules; pharmacology implicates a role for PKC and Ca2+ responses as m1 mAChR-mediated inhibition of GIRK channels is mimicked by PMA and Ca2+ ionophore. We have analyzed a series of mutant and chimeric channels suggesting that the GIRK4 subunit is capable of responding to G(q) signals and that the resulting current inhibition does not occur via phosphorylation of a canonical PKC site on the channel itself.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Bloqueadores dos Canais de Potássio , Canais de Potássio Corretores do Fluxo de Internalização , Receptores Muscarínicos/metabolismo , Sinalização do Cálcio , Clonagem Molecular , Condutividade Elétrica , Eletrofisiologia/métodos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fosforilação , Canais de Potássio/genética , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Sistemas do Segundo MensageiroRESUMO
Cardiac m2 muscarinic acetylcholine receptors reduce heart rate by coupling to heterotrimeric (alpha beta gamma) guanine nucleotide-binding (G) proteins that activate IKACh, an inward rectifier K+ channel (IRK). Activation of the GIRK subunit of IKACh requires G beta gamma subunits; however, the structural basis of channel regulation is unknown. To determine which sequences confer G beta gamma regulation upon IRKs, we generated chimeric proteins composed of GIRK and RB-IRK2, a related, G protein-insensitive channel. Importantly, a chimeric channel containing the hydrophobic pore region of RB-IRK2 joined to the amino and carboxyl termini of GIRK exhibited voltage- and receptor-dependent activation in Xenopus oocytes. Furthermore, carboxy-terminal sequences specific to this chimera and GIRK bound G beta gamma subunits in vitro. Thus, G beta gamma may regulate IRKs by interacting with sequences adjacent to the putative channel pore.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Eletrofisiologia , Proteínas de Ligação ao GTP/química , Dados de Sequência Molecular , Oócitos/fisiologia , Canais de Potássio/química , Canais de Potássio/ultraestrutura , Ligação Proteica/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Xenopus laevisRESUMO
Similar 23-base-pair (bp) direct repeats occur at the ends of two adjacent but noncontiguous T-DNAs, TL and TR (left and right T-DNA), in the tumor-inducing plasmid pTiA6NC. Thus, three border repeats lie right and one lies left of TL, which carries the genes needed for tumor maintenance. To determine whether T-DNA transfer and integration (subsequently called T-DNA transmission) require sequences in addition to the 23-bp border repeat, we constructed a deletion removing the three potential TL right borders (the TL right border and both TR borders). Since this deletion severely attenuated virulence, we reintroduced restriction fragments containing the TL right border repeat at a new location to the right of TL and tested their ability to restore virulence. Fragments that carried the border repeat flanked by at least 67 bp of wild-type Ti plasmid sequences on the left and 1035 bp on the right restored virulence completely. Smaller fragments restored virulence significantly but not fully, even though the border repeat remained intact. Therefore, T-region sequences flanking the border repeat in the fully active fragments stimulated T-DNA integration. Fragments that restored virulence fully when inserted in the wild-type orientation stimulated virulence only slightly in the opposite orientation. Thus, the right border sequence promotes T-DNA transfer and integration best in one direction.
Assuntos
Tumores de Planta/microbiologia , Plasmídeos , Rhizobium/genética , Sequência de Bases , Deleção Cromossômica , DNA Bacteriano/genética , Regulação da Expressão Gênica , Plantas , Sequências Repetitivas de Ácido NucleicoRESUMO
The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.
Assuntos
Aminoácidos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Aminoácidos/química , Animais , Cálcio/metabolismo , Linhagem Celular , Eletroquímica , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Receptores Muscarínicos/química , Receptores Muscarínicos/genética , Fosfolipases Tipo C/metabolismo , Xenopus laevisRESUMO
Several types of transmembrane receptors regulate cellular responses through the activation of phospholipase C-mediated Ca2+ release from intracellular stores. In non-excitable cells, the initial Ca2+ release is typically followed by a prolonged Ca2+ influx phase that is important for the regulation of several Ca2+-sensitive responses. Here we describe an agonist concentration-dependent mechanism by which m3 muscarinic acetylcholine receptors (mAChRs) differentially regulate the magnitude of the release and influx components of a Ca2+ response. In transfected Chinese hamster ovary cells expressing m3 mAChRs, doses of the muscarinic agonist carbachol ranging from 100 nM to 1 mM evoked Ca2+ release responses of increasing magnitude; maximal Ca2+ release was elicited by the highest carbachol concentration. In contrast, Ca2+ influx was maximal when m3 mAChRs were activated by moderate doses (1-10 microM) of carbachol, but substantially reduced at higher agonist concentrations. Manipulation of the membrane potential revealed that the carbachol-induced Ca2+ influx phase was diminished at depolarized potentials. Importantly, carbachol doses above 10 microM were found to couple m3 mAChRs to the activation of an inward, monovalent cation current resulting in depolarization of the cell membrane and a selective decrease in the influx, but not release, component of the Ca2+ response. These studies demonstrate, in one experimental system, a mechanism by which a single subtype of G-protein-coupled receptor can utilize the information encoded in the concentration of an agonist to generate distinct intracellular Ca2+ signals.
Assuntos
Cálcio/metabolismo , Cátions Monovalentes/metabolismo , Canais Iônicos/metabolismo , Receptores Muscarínicos/fisiologia , Animais , Células CHO , Carbacol/farmacologia , Cátions Bivalentes , Cricetinae , Relação Dose-Resposta a Droga , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Receptor Muscarínico M3 , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the G protein-coupled m1 muscarinic acetylcholine receptor potently suppresses a cloned delayed rectifier K+ channel through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the K+ channel. Furthermore, analysis of neuroblastoma cells revealed that a similar tyrosine kinase-dependent pathway links endogenous G protein-coupled receptors to suppression of the native RAK channel. These results suggest a novel mechanism by which neurotransmitters and hormones may regulate a specific type of K+ channel that is widely expressed in the mammalian brain and heart.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Canais de Potássio/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Muscarínicos/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Embrião de Mamíferos , Embrião não Mamífero , Feminino , Proteínas de Ligação ao GTP/biossíntese , Genisteína , Humanos , Isoflavonas/farmacologia , Rim , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Neuroblastoma , Oócitos/fisiologia , Bloqueadores dos Canais de Potássio , Canais de Potássio/biossíntese , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Muscarínicos/biossíntese , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas , XenopusRESUMO
Previous studies have shown that a single type of transmembrane receptor is able to regulate multiple effectors through the activation of heterotrimeric G proteins. For example, the m2 muscarinic acetylcholine receptor (mAChR) expressed in Chinese hamster ovary (CHO) cells inhibits adenylyl cyclase, stimulates phospholipase C-dependent intracellular Ca2+ release, and activates phospholipase A2 through pertussis toxin-sensitive G proteins. However, it is unclear whether multiple effector enzymes can be regulated by one type of heterotrimeric G protein within a single cell. To investigate this question, we constructed a derivative of G alpha i3 (termed G alpha i3 C > S) in which the carboxyl-terminal cysteine residue, the site for pertussis toxin modification, was changed to a serine. Following pertussis toxin treatment of transfected CHO cells expressing the m2 mAChR, we found that the G alpha i3 C > S protein underwent guanine nucleotide exchange in response to the muscarinic agonist carbachol, while the m2 mAChR failed to activate the endogenous G alpha i2 and G alpha i3 proteins. Moreover, coupling of heterotrimeric G proteins containing G alpha i3 C > S to the m2 mAChR resulted in pertussis toxin-resistant inhibition of adenylyl cyclase, stimulation of phospholipase C-induced intracellular Ca2+ release, and phospholipase A2-mediated arachidonic acid release. Therefore, these studies provide conclusive evidence that heterotrimeric G proteins containing just G alpha i3 can regulate multiple effector enzymes within the same cell type.
Assuntos
Inibidores de Adenilil Ciclases , Proteínas de Ligação ao GTP/metabolismo , Fosfolipases A/metabolismo , Fosfolipases Tipo C/metabolismo , Toxina Adenilato Ciclase , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Ativação Enzimática , Proteínas de Ligação ao GTP/química , Toxina Pertussis , Fosfolipases A2 , Receptores Muscarínicos/fisiologia , Transdução de Sinais , Fatores de Virulência de Bordetella/farmacologiaRESUMO
During crown gall tumorigenesis a specific segment of the Agrobacterium tumefaciens tumour-inducing (Ti) plasmid, the T-DNA, integrates into plant nuclear DNA. Similar 23-bp direct repeats at each end of the T region signal T-DNA borders, and T-DNA transmission (transfer and integration) requires the right-hand direct repeat. A chemically synthesized right border repeat in its wild-type orientation promotes T-DNA transmission at a low frequency; Ti plasmid sequences which normally flank the right repeat greatly stimulate the process. To identify flanking sequences required for full right border activity, we tested the activity of a border repeat surrounded by different amounts of normal flanking sequences. Efficient T-DNA transmission required a conserved sequence (5' TAAPuTPy-CTGTPuT-TGTTTGTTTG 3') which lies to the right of the two known right border repeats. In either orientation, a synthetic oligonucleotide containing this conserved sequence greatly stimulated the activity of a right border repeat, and a deletion removing 15 bp from the right end of this sequence destroyed it stimulatory effect. Thus, wild-type T-DNA transmission required both the 23-bp right border repeat and a conserved flanking sequence which we call overdrive.
Assuntos
DNA Bacteriano/genética , Elementos Facilitadores Genéticos/genética , Genes Bacterianos/genética , Plasmídeos Indutores de Tumores em Plantas/genética , Agrobacterium tumefaciens/genética , Sequência de Bases , Dados de Sequência MolecularRESUMO
Muscarinic receptor-induced changes in the activities of phospholipase D (PLD) and of phosphoinositide-phospholipase C (PI-PLC) were investigated in human embryonic kidney (HEK) cells transfected with, and stably expressing, the human m1, m2, m3, and m4 mAChR subtypes, respectively. PLD and PI-PLC activities in these four transfected cell lines as well as in nontransfected cells were measured by the formation of [3H]phosphatidylethanol [( 3H]PEt) and [3H]inositol phosphates [( 3H]IP) after labeling cellular phospholipids with [3H]oleic acid and [3H]inositol. The muscarinic receptor agonist carbachol had no significant effects on [3H]PEt and [3H]IP formation in nontransfected HEK cells. In cells expressing the m1 or m3 receptors carbachol (1 mM; in the presence of 400 mM ethanol and 10 mM lithium chloride) caused the formation of [3H]PEt of about 12,000 cpm/mg protein (basal PEt formation was not measurable) and increased [3H]IP formation by 20,000-30,000 cpm/mg (a 7-10-fold increase over basal levels). The EC50 values (0.3-1.5 microM) were similar for both effects and both mAChR subtypes. In contrast, in cells expressing m2 or m4 receptor subtypes the magnitude of [3H]PEt (about 4,000 cpm/mg protein) or [3H]IP (3,000-4,000 cpm/mg) formation was much smaller and the EC50 values (20-40 microM) much higher than for the m1 and m3 receptors. Neomycin (1 mM) inhibited the m1 and m3 receptor-mediated production of IP by 50%, whereas the PEt formation was attenuated by 20% in the same cells. We conclude that activation of all of the four mAChR subtypes, although with different efficiencies, can stimulate PLD. The m1 and m3 receptor-mediated stimulation of the PLD may be at least partially independent of the PI-PLC stimulation.
Assuntos
Acetilcolina/metabolismo , Glicerofosfolipídeos , Fosfolipase D/metabolismo , Receptores Muscarínicos/metabolismo , Transfecção , Carbacol/farmacologia , Linhagem Celular , Ativação Enzimática , Humanos , Fosfatos de Inositol/biossíntese , Rim/citologia , Rim/efeitos dos fármacos , Rim/enzimologia , Ácidos Fosfatídicos/biossíntese , Receptores Muscarínicos/genéticaRESUMO
Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.