RESUMEN
One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.
Asunto(s)
Arrestinas/fisiología , Proteínas de Unión al ADN/genética , Proteínas/metabolismo , Factores de Transcripción/genética , Transcripción Genética/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Arrestinas/metabolismo , Línea Celular , Proteínas Dishevelled , Humanos , Factor de Unión 1 al Potenciador Linfoide , Ratones , Fosfoproteínas , Fosforilación , Unión Proteica , beta-ArrestinasRESUMEN
Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.
Asunto(s)
Proteínas Portadoras/metabolismo , Etilmaleimida/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Transporte Vesicular , Animales , Sitios de Unión , Western Blotting , Células COS , Proteínas Portadoras/química , Línea Celular , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Immunoblotting , Isoproterenol/farmacología , Proteínas Luminiscentes/metabolismo , Mutación , Proteínas Sensibles a N-Etilmaleimida , Pruebas de Precipitina , Propranolol/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta 2/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Anclaje a la Quinasa A , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Quinasas de Receptores Adrenérgicos betaRESUMEN
The beta(1)-adrenergic receptor (beta(1)AR) is the most abundant subtype of beta-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal beta(1)AR-interacting proteins, we screened a rat brain cDNA library using the beta(1)AR carboxyl terminus (beta(1)AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the beta(1)AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the beta(1)AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length beta(1)AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between beta(1)AR and PSD-95 is mediated by the last few amino acids of the beta(1)AR, and mutation of the beta(1)AR carboxyl terminus eliminated the binding and disrupted the co-localization of the beta(1)AR and PSD-95 in cells. Agonist-induced internalization of the beta(1)AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the beta(1)AR or beta(1)AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the beta(1)AR and N-methyl-d-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific beta(1)AR binding partner that modulates beta(1)AR function and facilitates physical association of the beta(1)AR with synaptic proteins, such as the N-methyl-d-aspartate receptors, which are known to be regulated by beta(1)AR stimulation.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Encéfalo/metabolismo , Células COS , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Homólogo 4 de la Proteína Discs Large , Biblioteca de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas del Tejido Nervioso/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Transporte de Proteínas , Ratas , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , TransfecciónRESUMEN
Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Agonistas de Receptores Adrenérgicos beta 1 , Animales , Bovinos , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Prolina/metabolismo , Unión Proteica , Receptores Adrenérgicos beta 1/química , Dominios Homologos srcRESUMEN
The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].
Asunto(s)
Secuencia de Consenso/genética , Cisteína/metabolismo , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/metabolismo , Transportadores de Anión Orgánico , Compuestos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/farmacología , 4-Cloromercuribencenosulfonato/metabolismo , 4-Cloromercuribencenosulfonato/farmacología , Aminas/metabolismo , Sustitución de Aminoácidos , Transporte Biológico/efectos de los fármacos , Permeabilidad de la Membrana Celular , Colina/metabolismo , Cisteína/genética , Proteínas de Escherichia coli , Etilmaleimida/metabolismo , Etilmaleimida/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Cinética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Moduladores del Transporte de Membrana , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Mesilatos/metabolismo , Mesilatos/farmacología , Modelos Biológicos , Poliaminas/metabolismo , Conformación Proteica , Especificidad por Sustrato , Reactivos de Sulfhidrilo/química , Reactivos de Sulfhidrilo/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The Escherichia coli gamma-aminobutyric acid permease (GabP) is a plasma membrane protein from the amine-polyamine-choline (APC) superfamily. On the basis of hydropathy analysis, transporters from this family are thought to contain 12, 13 or 14 transmembrane domains. We have experimentally analysed the topography of GabP by using the cytoplasmically active LacZ (beta-galactosidase) and the periplasmically active PhoA (alkaline phosphatase) as complementary topological sensors. The enzymic activities of 32 GabP-LacZ hybrids and 43 GabP-PhoA hybrids provide mutually reinforcing lines of evidence that the E. coli GabP contains 12 transmembrane segments that traverse the membrane in a zig-zag fashion with both N- and C-termini facing the cytoplasm. Interestingly, the resulting model predicts that the functionally important 'consensus amphipathic region' (CAR) [Hu and King (1998) Biochem. J. 330, 771-776] is at least partly membrane-embedded in many amino acid transporters from bacteria and fungi, in contrast with the apparent situation in mouse cationic amino acid transporters (MCATs), in which this kinetically significant region is thought to be fully cytoplasmic [Sophianopoulou and Diallinas (1995) FEMS Microbiol. Rev. 16, 53-75]. To the extent that conserved domains serve similar functions, the resolution of this topological disparity stands to have family-wide implications on the mechanistic role of the CAR. The consensus transmembrane structure derived from this analysis of GabP provides a foundation for predicting the topological disposition of the CAR and other functionally important domains that are conserved throughout the APC transporter superfamily.
Asunto(s)
Proteínas Portadoras/química , Escherichia coli/química , Proteínas de la Membrana/química , Transportadores de Anión Orgánico , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/química , Cartilla de ADN , Proteínas de Escherichia coli , Proteínas Transportadoras de GABA en la Membrana Plasmática , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
gab permease (GabP) is the exclusive mediator of 4-aminobutyrate (GABA) transport across the Escherichia coli plasma membrane. Helix 8 and a portion of the adjoining cytoplasmic region (loop 8-9) constitute the GabP "consensus amphipathic region" (CAR), a potential channel-forming domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily. Upon the polar surface of the CAR, all known gab permeases display a "signature cysteine" not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting specificity (kcat/Km) to the translocation reaction. Here we show that among the five cysteine residues in the E. coli GabP, only Cys-300, the signature cysteine, can restore wild type properties to the Cys-less GabP mutant. We conclude (i) from partial reaction studies (equilibrium exchange, counterflow) that rapid translocation of the GABA binding site from one side of the membrane to the other is greatly facilitated by Cys-300 and (ii) from pharmacological studies that loss of Cys-300 has little effect on the affinity that GabP exhibits for a structurally diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic acid, and cis-4-aminocrotonic acid) of competitive ligands. These results raise the possibility that other GABA transporters might rely analogously upon conserved cysteine residues positioned within the amphipathic helix 8 and loop 8-9 regions.
Asunto(s)
Proteínas Portadoras/química , Cisteína/química , Escherichia coli/fisiología , Proteínas de la Membrana/química , Transportadores de Anión Orgánico , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión/fisiología , Transporte Biológico/fisiología , Secuencia Conservada/genética , Proteínas de Escherichia coli , Proteínas Transportadoras de GABA en la Membrana Plasmática , Proteínas de Transporte de Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Ácido gamma-Aminobutírico/farmacocinéticaRESUMEN
The gab permease (GabP) catalyses transport of GABA (4-aminobutyrate) into Escherichia coli. Although GabP can recognize and transport many GABA analogues that exhibit activity at GABAergic synapses in the nervous system, the protein domains responsible for these transport and ligand recognition properties have not been studied. Here we report that an amphipathic domain extending through putative transmembrane helix 8 and into the adjoining cytoplasmic region (loop 8-9) contains a critical 20 residue zone within which mutagenesis of polar amino acids has a deleterious effect on [3H]GABA transport activity. This functionally important amphipathic domain is found to be highly conserved in the many APC family transporters that are homologous to GabP. And even though members of the GAT family of GABA transporters from the animal nervous system are not homologous to GabP, an analogous amphipathic structure is found in their loop 8-9 region. These results and observations suggest: (1) that the consensus amphipathic region (CAR) in the putative helix 8 and loop 8-9 region of GabP has functional significance, and (2) that nature has repeatedly used this CAR in transporters from bacteria to mammals.