RESUMO
Several different molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G(i/o)). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an approximately 100 - 1000 fold lower potency compared with the different molecular species of PA. RT - PCR using gene-specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/farmacologia , Ácidos Fosfatídicos/farmacologia , Receptores de Superfície Celular/agonistas , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Rim/citologia , Rim/embriologia , Rim/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA/genética , RNA/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The formation of complexes between growth factor receptors and members of a family of G-protein-coupled receptors whose natural ligands are S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) represents a new signalling entity. This receptor complex allows for integrated signalling in response to growth factor and/or S1P/LPA and provides a mechanism for more efficient activation (due to integrated close-proximity signalling from both receptor classes) of the p42/p44 MAPK (mitogen-activated protein kinase) pathway. This article provides information on the molecular events at the interface between receptor tyrosine kinases and S1P/LPA receptors. Examples include the PDGF (platelet-derived growth factor)-induced tyrosine phosphorylation of G(i)alpha, released upon S1P(1) receptor activation, which is required for initiation of the p42/p44 MAPK pathway. Critical to this event is the formation of endocytic vesicles containing functionally active PDGFbeta receptor-S1P(1) receptor complexes, which are internalized and relocated with components of the p42/p44 MAPK pathway. We also report examples of cross-talk signal integration between the Trk A (tropomyosin receptor kinase A) receptor and the LPA(1) receptor in terms of the NGF (nerve growth factor)-dependent regulation of the p42/p44 MAPK pathway. NGF induces recruitment of the LPA(1) receptor to the nucleus (delivery might be Trk A-dependent), whereupon the LPA(1) receptor may govern gene expression via novel nuclear signalling processes.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linhagem Celular , Humanos , Fator de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LisofosfolipídeosRESUMO
The inhibitory gamma subunits of the retinal rod and cone photoreceptor type 6 retinal cyclic guanosine monophosphate phosphodiesterase (PDEgamma) are expressed in non-retinal tissues. Here, we show that PDEgamma interacts with the G-protein-coupled receptor kinase 2 signaling system to regulate the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. This is based upon several lines of evidence. First, the transfection of cells with an antisense rod PDEgamma plasmid construct, which reduced endogenous rod PDEgamma expression, ablated the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase. Second, the transfection of cells with recombinant rod or cone PDEgamma and/or G-protein-coupled receptor kinase 2 increased the stimulation of p42/p44 mitogen-activated protein kinase by epidermal growth factor or thrombin. In contrast, a G-protein-coupled receptor kinase 2 phosphorylation-resistant rod PDEgamma mutant failed to increase the epidermal growth factor- or thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase and, in fact, functioned as a dominant negative. Thrombin also stimulated the association of endogenous rod PDEgamma with dynamin II, which was increased in cells transfected with rod PDEgamma or G-protein-coupled receptor kinase 2. Dynamin II plays a critical role in regulating endocytosis of receptor signal complexes required for activation of p42/p44 mitogen-activated protein kinase. Therefore, PDEgamma may have an important role in promoting endocytosis of receptor signal complexes leading to the activation of p42/p44 mitogen-activated protein kinase. We conclude that PDEgamma is an entirely novel intermediate regulating mitogenic signaling from both receptor tyrosine kinase and G-protein-coupled receptors in human embryonic kidney 293 cells.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Retina/enzimologia , Transdução de Sinais , 3',5'-GMP Cíclico Fosfodiesterases/química , 3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Dinaminas , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Rim/citologia , Rim/embriologia , Rim/enzimologia , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidato Fosfatase/metabolismo , Receptores de Superfície Celular/fisiologia , Linhagem Celular , Membrana Celular/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Isoenzimas/metabolismo , Rim , Cinética , Lisofosfolipídeos/farmacologia , Proteína Quinase 3 Ativada por Mitógeno , Dibutirato de 12,13-Forbol/farmacologia , Ácidos Fosfatídicos/farmacologia , Fosfolipase D/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.