RESUMEN
BACKGROUND: We previously reported that the guanine tri-phosphate-binding proteins (G) beta and gamma are both localized in the nucleus, in addition to their expected cytoplasmic/plasma membrane localization. These proteins, as a heterodimeric complex, suppress glucocorticoid response element-mediated transcriptional activity of the glucocorticoid receptor through direct physical interactions between Gbeta and the glucocorticoid receptor. MATERIALS AND METHODS: As Ggamma is prenylated at a cysteine residue in its C-terminal portion, and as this post-translational modification is required for many of the known Gbeta/Ggamma activities, we examined the effect of its absence or diminution on Gbeta/Ggamma-induced suppression of glucocorticoid receptor-induced transcriptional activity. RESULTS: In a functional reporter assay, Ggamma2C68S, which is defective at the prenylation site, was more potent than the wild-type Ggamma2 at increasing Gbeta2-induced suppression of glucocorticoid receptor transactivation. Interestingly, the enhanced green fluorescent protein fusion of this mutant Ggamma2 was localized preferentially in the nucleus, while it was absent from the plasma membrane. Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that abrogates the prenylation of Ggamma, shifted the subcellular localization of enhanced green fluorescence protein-fused Ggamma2 and Gbeta2 from the cytoplasm/plasma membrane to the nucleus and further suppressed glucocorticoid receptor-induced transcriptional activity. CONCLUSIONS: These findings indicate that not only is the natural covalent addition of the prenyl residue to Ggamma unnecessary for the transcriptional suppression induced by Gbeta/Ggamma on the glucocorticoid receptor, but rather helps retain the Gbeta/Ggamma complex away from the nucleus decreasing its antiglucocorticoid actions.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Receptores de Glucocorticoides/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HCT116 , Humanos , Microscopía Confocal/métodos , Mutación , Prenilación de Proteína/genética , Prenilación de Proteína/fisiología , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , TransfecciónRESUMEN
Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Anclaje a la Quinasa A , Células Cultivadas , AMP Cíclico/metabolismo , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Nucleótidos de Guanina/metabolismo , Humanos , Riñón/citología , Masculino , Modelos Biológicos , Unión Proteica , Subunidades de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor kappa B (NF-kappa B) and secretion of interleukin-8, and this response involves activation of G alpha(13) and RhoA. The induced expression of a NF-kappa B luciferase reporter was partially reduced by pertussis toxin and the G beta gamma scavenger transducin, and enhanced by co-expression of G alpha(13) and to a lesser extent, G alpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kappa B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G alpha(13)-RhoA pathway in KSHV-GPCR-mediated NF-kappa B activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kappa B activation by KSHV-GPCR, and by a constitutively active G alpha(13)(Q226L). KSHV-GPCR-induced NF-kappa B activation is accompanied by increased secretion of IL-8, a function mimicked by the activated G alpha(13) but not by an activated G alpha(q)(Q209L). These results suggest coupling of KSHV-GPCR to the G alpha(13)-RhoA pathway in addition to other G proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Herpesvirus Humano 6/metabolismo , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Receptores de Superficie Celular/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Células HeLa , Humanos , Sarcoma de Kaposi/virología , Transducción de SeñalRESUMEN
The G alpha subunit of G(12) protein, one of the heterotrimeric G proteins, regulates diverse and complex cellular responses by transducing signals from the cell surface, presumably involving more than one downstream effector. Yeast two-hybrid screening of a human testis cDNA library identified a large fragment of Hsp90 as a protein that interacted with G alpha(12). The interaction between G alpha(12) and Hsp90 was further substantiated by a co-immunoprecipitation technique. We have determined that Hsp90 is not required for the interaction of G alpha(12) with its binding partners, p115(RhoGEF) and the G beta subunit. Importantly, Hsp90 is required for G alpha(12)-induced serum response element activation, cytoskeletal changes, and mitogenic response. Closely related to G alpha(12), the G alpha(13) subunit did not interact with Hsp90 and did not require functional Hsp90 for serum response element activation. Thus, our results identify a novel signaling module of G alpha(12) and Hsp90.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Transducción de Señal , Células 3T3 , Animales , Células COS , Citoesqueleto/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Ratones , Microscopía Fluorescente , Unión ProteicaRESUMEN
The regulator of G-protein signaling (RGS) negatively regulates the alpha subunit of G proteins by accelerating their intrinsic guanosine triphosphatase (GTPase) activity. Here are reported the isolation and characterization of a novel mouse RGS, termed RGS18, which is a new member of RGS subfamily B. Northern blot analysis showed that RGS18 messenger RNA was detected predominantly in spleen and hematopoietic cells, and immunohistochemical studies demonstrated that RGS18 was expressed in megakaryocytes, platelets, granulocytes/monocytes, and, weakly, in hematopoietic stem cells, but not in lymphocytes or erythrocytes. Although various subcellular localizations of RGS have been reported, RGS18 was found to be localized in cytoplasm in megakaryocytes. In vitro binding assays of RGS18 with megakaryocyte cell lysates with or without AlF(4)(-) treatment demonstrated that RGS18 specifically binds to 2 alpha subunits of the G protein, Galphai and Galphaq. Furthermore, RGS18 clearly exhibited GTPase-activating protein (GAP) activity for Galphai and Galphaq but not for Galphas or Galpha12. In addition, chemokine stromal-derived factor 1 (SDF-1), which has been reported to stimulate megakaryocyte colony formation in the presence of thrombopoietin, affected the binding of RGS18 to Galphai but not to Galphaq. Therefore, the newly isolated RGS18 turned out to be a new member of the RGS family bearing GAP activity for Galphai, which might be stimulated by SDF-1 in megakaryocytes, as well as for Galphaq. Thus, RGS18 may play an important role in proliferation, differentiation, and/or migration of megakaryocytes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Compuestos de Aluminio/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Plaquetas/química , Northern Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Citoplasma/química , Femenino , Fluoruros/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Granulocitos/química , Células Madre Hematopoyéticas/química , Inmunohistoquímica , Megacariocitos/química , Megacariocitos/ultraestructura , Ratones , Datos de Secuencia Molecular , Monocitos/química , Proteínas RGS , ARN Mensajero/análisis , Bazo/químicaRESUMEN
RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.
Asunto(s)
Reguladores de Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Transducción de Señal , Animales , Bovinos , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/análisis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Estructura Terciaria de Proteína , Factores de Intercambio de Guanina Nucleótido Rho , Quinasas de Receptores Adrenérgicos betaRESUMEN
G protein-coupled inward rectifier K(+) channels (GIRK channels) are activated directly by the G protein betagamma subunit. The crystal structure of the G protein betagamma subunits reveals that the beta subunit consists of an N-terminal alpha helix followed by a symmetrical seven-bladed propeller structure. Each blade is made up of four antiparallel beta strands. The top surface of the propeller structure interacts with the Galpha subunit. The outer surface of the betagamma torus is largely made from outer beta strands of the propeller. We analyzed the interaction between the beta subunit and brain GIRK channels by mutating the outer surface of the betagamma torus. Mutants of the outer surface of the beta(1) subunit were generated by replacing the sequences at the outer beta strands of each blade with corresponding sequences of the yeast beta subunit, STE4. The mutant beta(1)gamma(2) subunits were expressed in and purified from Sf9 cells. They were applied to inside-out patches of cultured locus coeruleus neurons. The wild type beta(1)gamma(2) induced robust GIRK channel activity with an EC(50) of about 4 nm. Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels. However, the ability of D1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta(1)gamma(2). As to the activities to stimulate phospholipase Cbeta(2), D1 was more potent and CD2 was less potent than the wild type beta(1)gamma(2). Additionally we tested four beta(1) mutants in which mutated residues are located in the top Galpha/beta interacting surface. Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels. These results suggest that the outer surface of blade 1 and blade 2 of the beta subunit might specifically interact with GIRK and that the beta subunit interacts with GIRK both over the outer surface and over the top Galpha interacting surface.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP , Proteínas de Unión al GTP Heterotriméricas/fisiología , Locus Coeruleus/fisiología , Canales de Potasio/fisiología , Proteínas de Saccharomyces cerevisiae , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Bovinos , Línea Celular , Membrana Celular/fisiología , Células Cultivadas , Activación Enzimática , Proteínas Fúngicas/fisiología , Proteínas de Unión al GTP Heterotriméricas/química , Isoenzimas/metabolismo , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Fosfolipasa C beta , Canales de Potasio/química , Estructura Secundaria de Proteína , Subunidades de Proteína , Ratas , Ratas Long-Evans , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/fisiología , Spodoptera , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
The outcome of the first stage of planetary formation, which is characterized by ballistic agglomeration of preplanetary dust grains due to Brownian motion in the free molecular flow regime of the solar nebula, is still somewhat speculative. We performed a microgravity experiment flown onboard the space shuttle in which we simulated, for the first time, the onset of free preplanetary dust accumulation and revealed the structures and growth rates of the first dust agglomerates in the young solar system. We find that a thermally aggregating swarm of dust particles evolves very rapidly and forms unexpected open-structured agglomerates.
Asunto(s)
Modelos Teóricos , Planetas , IngravidezRESUMEN
Regulator of G protein signaling (RGS) proteins are a family of approximately 20 proteins that negatively regulate signaling through heterotrimeric G protein-coupled receptors. The RGS proteins act as GTPase-activating proteins (GAPs) for certain Galpha subunits and as effector antagonists for Gqalpha. Mouse RGS14 encodes a 547-amino-acid protein with an N-terminal RGS domain, which is highly expressed in lymphoid tissues. In this study, we demonstrate that RGS14 is a GAP for Gialpha subfamily members and it attenuates interleukin-8 receptor-mediated mitogen-activated protein kinase activation. However, RGS14 does not exhibit GAP activity toward Gsalpha or Gqalpha nor does it regulate Gsalpha- or Gqalpha-mediated signaling pathways. Although RGS14 does not act as a GAP for G12/13alpha, it impairs c-fos serum response element activation induced by either a constitutively active mutant of G13alpha (G13alphaQ226L) or by carbachol stimulation of muscarinic type 1 receptors. An RGS14 mutant (EN92/93AA), which does not block Gialpha-linked signaling, also inhibits serum response element activation. RGS14 localizes predominantly in the cytosol, but it can be recruited to membranes by expression of G13alphaQ226L. Although RGS14 is constitutively expressed in lymphoid cells, agents that activate B or T lymphocytes further enhance its levels. Taken together, our results suggest that signals generated after lymphocyte activation may via RGS14 directly impinge on Gialpha- or G13alpha-mediated cellular processes in lymphocytes, such as adhesion and migration.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas RGS/metabolismo , Animales , Linfocitos B/metabolismo , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Expresión Génica , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas RGS/genética , Transducción de Señal , Fracciones Subcelulares , Linfocitos T/metabolismoRESUMEN
G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Quinasa 2 de Adhesión Focal , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Genes Reporteros , Células HeLa , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Luciferasas/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transcripción Genética , Transfección , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13). Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de la Membrana/metabolismo , Células 3T3 , Animales , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Ratones , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Transducción de Señal , LevadurasRESUMEN
Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.
Asunto(s)
Linfocitos B/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas/fisiología , Proteínas RGS , Transducción de Señal/inmunología , Animales , Linfocitos B/inmunología , Células COS , Regulación hacia Abajo/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/fisiología , Humanos , Células Jurkat , Células K562 , Unión Proteica/inmunología , Biosíntesis de Proteínas , Ratas , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/fisiología , Células Tumorales CultivadasRESUMEN
G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas RGS/metabolismo , Compuestos de Aluminio/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Unión Competitiva , Encéfalo/metabolismo , Células COS , Bovinos , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática , Fluoruros/farmacología , Quinasa 3 del Receptor Acoplado a Proteína-G , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP/genética , Humanos , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Fosfolipasa C beta , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas RGS/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Extractos de Tejidos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Quinasas de Receptores Adrenérgicos betaRESUMEN
An effector candidate for G protein action, GRIN1, was identified by screening a cDNA expression library with phosphorylated GTPgammaS-G(z)alpha as a probe. GRIN1 is a novel protein without substantial homology to known protein domains. It is expressed largely in brain and binds specifically to activated G(z)alpha, G(o)alpha, and G(i)alpha through its carboxyl-terminal region. The protein KIAA0514 (GRIN2) is homologous to GRIN1 at its carboxyl terminus and also binds to activated G(o)alpha. Both GRIN1 and G(o)alpha are membrane-bound proteins that are enriched in the growth cones of neurites. Coexpression of GRIN1 or GRIN2 with activated G(o)alpha causes formation of a network of fine processes in Neuro2a cells, suggesting that these pathways may function downstream of G(o)alpha to control growth of neurites.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas del Tejido Nervioso , Receptores de N-Metil-D-Aspartato , Alineación de SecuenciaRESUMEN
The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Sitios de Unión , Activación Enzimática , Proteínas de Unión al GTP/genética , Humanos , Modelos Moleculares , Mutagénesis , Fosfolipasa C beta , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to modulate various cellular processes. The direct downstream effectors of G proteins consist of the signalling molecules that are activated by their physical interactions with a G alpha or Gbetagamma subunit. Effectors that interact directly with G alpha12 G proteins have yet to be identified. Here we show that G alpha12 binds directly to, and stimulates the activity of, Bruton's tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo. G alpha12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif, that is present in both Btk and Gap1m. Our results are, to our knowledge, the first to identify direct effectors for G alpha12 and to show that there is a direct link between heterotrimeric and monomeric G proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfoproteínas , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Sitios de Unión , Proteínas Sanguíneas/química , Células COS , Línea Celular , Clonación Molecular , Activación Enzimática , Escherichia coli , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Proteínas Tirosina Quinasas/química , Receptores de Tromboxanos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Proteínas Activadoras de ras GTPasaRESUMEN
We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J., Song, X., and Ross, E. M. (1997) J. Biol. Chem. 272, 5732-5740) and a member of the RGS family of G protein GAPs. Its sequence is 83% identical to RET-RGS1 (except its N-terminal extension) and 56% identical to GAIP. Purified, recombinant RGSZ1, RET-RGS1, and GAIP each accelerated the hydrolysis of Galphaz-GTP over 400-fold with Km values of approximately 2 nM. RGSZ1 was 100-fold selective for Galphaz over Galphai, unusually specific among RGS proteins. Other enzymological properties of RGSZ1, brain Gz GAP, and RET-RGS1 were identical; GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Galphaz. RGSZ1, RET-RGS1, and GAIP thus define a subfamily of Gz GAPs within the RGS proteins. RGSZ1 has no obvious membrane-spanning region but is tightly membrane-bound in brain. Its regulatory activity in membranes depends on stable bilayer association. When co-reconstituted into phospholipid vesicles with Gz and m2 muscarinic receptors, RGSZ1 increased agonist-stimulated GTPase >15-fold with EC50 <12 nM, but RGSZ1 added to the vesicle suspension was <0.1% as active. RGSZ1, RET-RGS1, and GAIP share a cysteine string sequence, perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by Gz. Phosphorylation of Galphaz by protein kinase C inhibited the GAP activity of RGSZ1 and other RGS proteins, providing a mechanism for potentiation of Gz signaling by protein kinase C.
Asunto(s)
Encéfalo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Proteínas/química , Proteínas RGS , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Detergentes/farmacología , Activación Enzimática/fisiología , Proteínas Activadoras de GTPasa , Humanos , Liposomas/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Heterotrimeric G proteins, composed of alpha and betagamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free betagamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by betagamma, it is likely that the alpha subunit binding surfaces on betagamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact alpha in the recently published crystal structures of the alphabetagamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade 1. We mutated four residues on the top surface of beta (Hbeta1[L117A], Hbeta1[D228R], Hbeta1[D246S], and Hbeta1[W332A]) and two residues on the side of beta that contacts alpha (Hbeta1[N88A/K89A]). Each of the mutant proteins was able to form beta gamma dimers, but they differed in their ability to bind alpha and to activate phospholipase C beta2 (PLCbeta2), PLCbeta3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the alpha binding surface differentially affected PLCbeta2, PLCbeta3, and adenylyl cyclase II. Residues that affect PLCbeta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the alpha subunit, make many contacts on the top surface. None of the mutations affected the ability of betagamma to inhibit adenylyl cyclase I. We conclude that alpha, PLCbeta2, PLCbeta3, and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for alpha binding and for effector activation and differs even between closely related isoforms of the same effector.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Baculoviridae , Sitios de Unión/genética , Células COS , Dimerización , Activación Enzimática , Proteínas de Unión al GTP/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfolipasa C beta , Conformación Proteica , Spodoptera , Relación Estructura-ActividadRESUMEN
Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Compuestos de Aluminio/metabolismo , Secuencia de Aminoácidos , Animales , Fluoruros/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Factores de Intercambio de Guanina Nucleótido , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Proteínas/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de SeñalRESUMEN
Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.