RESUMEN
G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the ß(2)-adrenergic receptor (ß(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified ß(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated ß(2)AR pull-down, 242 proteins in the inverse agonist-activated ß(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the ß(2)AR-interacting proteins isolated was confirmed by Western blot; three known ß(2)AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known ß(2)AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of ß(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.
Asunto(s)
Proteínas Portadoras/metabolismo , Lipoproteínas HDL/metabolismo , Espectrometría de Masas , Proteómica , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Biología Computacional/métodos , Citosol/metabolismo , Inmunoprecipitación , Unión Proteica , Mapeo de Interacción de Proteínas , Ratas , Receptores Adrenérgicos beta 2/metabolismo , Reproducibilidad de los ResultadosRESUMEN
A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co-culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co-cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild-type and beta-adrenergic receptor (beta-AR) knockout cardiomyocytes and simultaneously exposed them with the beta-AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of beta-receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co-culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas de Cocultivo/métodos , Microelectrodos , Miocitos Cardíacos/citología , Animales , Animales Recién Nacidos , Células Cultivadas , Ratones , Miocitos Cardíacos/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores Adrenérgicos beta 2/inmunología , Receptores Acoplados a Proteínas G/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Reacciones Antígeno-Anticuerpo/inmunología , Western Blotting , Cristalización/métodos , Cristalografía , Epítopos/química , Epítopos/inmunología , Colorantes Fluorescentes/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Rodaminas/química , VacunaciónRESUMEN
G-protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G-protein-coupled receptors (GPCRs). All seven mammalian GRKs contain an N-terminal domain that is homologous to the regulator of G-protein signaling (RGS) family of proteins. The RGS domain of GRK2 has been shown to interact specifically with Galphaq family members. While the specificity and functional consequences of GRK/Galpha interaction remain somewhat poorly defined, GRK RGS homology (RH) domains likely function to provide specificity for GRK interaction with a particular Galpha subunit or GPCR/Galpha complex. Indeed, GRK2 binds Galphaq, alpha11, and alpha14, but not Galpha16, Galphas, Galphai, or Galpha(12/13), while the RGS domains of GRK5 and GRK6 do not bind Galpha(q/11), Galphas, Galphai, or Galpha(12/13). In this chapter we describe various in vitro and intact cell strategies that can be used to elucidate the function of GRK RH domains.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistemas de Mensajero Secundario/fisiología , Animales , Bioensayo/métodos , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Humanos , Fosfatos de Inositol/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G(q) subfamily of Galpha subunits couples GPCR activation to the enzymatic activity of phospholipase C-beta (PLC-beta). Regulators of G protein signaling (RGS) proteins bind to activated Galpha subunits, including Galpha(q), and regulate Galpha signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Galpha subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Galpha(q) attenuates PLC-beta activity. The RH domain of GRK2 interacts with Galpha(q/11) through a novel Galpha binding surface termed the "C" site. Here, molecular modeling of the Galpha(q).GRK2 complex and site-directed mutagenesis of Galpha(q) were used to identify residues in Galpha(q) that interact with GRK2. The model identifies Pro(185) in Switch I of Galpha(q) as being at the crux of the interface, and mutation of this residue to lysine disrupts Galpha(q) binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Galpha(q)-V240A, Galpha(q)-D243A, both residues within Switch III, and Galpha(q)-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Galpha(q)-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Galpha(q)-R183C. Interestingly, the model also predicts that residues in the helical domain of Galpha(q) interact with GRK2. In fact, the mutants Galpha(q)-K77A, Galpha(q)-L78D, Galpha(q)-Q81A, and Galpha(q)-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Galpha(q)-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Galpha(q) interaction rely on contacts with distinct regions and different Switch I residues in Galpha(q).
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Sitios de Unión , Línea Celular , Dimerización , Electroforesis en Gel de Poliacrilamida , Quinasa 2 del Receptor Acoplado a Proteína-G , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Fosfatos de Inositol/química , Isoenzimas/metabolismo , Lisina/química , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Fosfolipasa C beta , Fosforilación , Plásmidos/metabolismo , Mutación Puntual , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Fosfolipasas de Tipo C/metabolismo , Quinasas de Receptores Adrenérgicos betaRESUMEN
Regulators of G protein signaling (RGS) proteins bind to active G alpha subunits and accelerate the rate of GTP hydrolysis and/or block interaction with effector molecules, thereby decreasing signal duration and strength. RGS proteins are defined by the presence of a conserved 120-residue region termed the RGS domain. Recently, it was shown that the G protein-coupled receptor kinase 2 (GRK2) contains an RGS domain that binds to the active form of G alpha(q). Here, the ability of GRK2 to interact with other members of the G alpha(q) family, G alpha(11), G alpha(14), and G alpha(16), was tested. The signaling of all members of the G alpha(q) family, with the exception of G alpha(16), was inhibited by GRK2. Immunoprecipitation of full-length GRK2 or pull down of GST-GRK2-(45-178) resulted in the detection of G alpha(q), but not G alpha(16), in an activation-dependent manner. Moreover, activated G alpha(16) failed to promote plasma membrane (PM) recruitment of a GRK2-(45-178)-GFP fusion protein. Assays with chimeric G alpha(q)(-)(16) subunits indicated that the C-terminus of G alpha(q) mediates binding to GRK2. Despite showing no interaction with GRK2, G alpha(16) does interact with RGS2, in both inositol phosphate and PM recruitment assays. Thus, GRK2 is the first identified RGS protein that discriminates between members of the G alpha(q) family, while another RGS protein, RGS2, binds to both G alpha(q) and G alpha(16).
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas Fluorescentes Verdes , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Proteínas Luminiscentes/genética , Familia de Multigenes/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Transporte de Proteínas/genética , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transfección , Quinasas de Receptores Adrenérgicos betaRESUMEN
G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G alpha(q/ll) and inhibiting G alpha(q) stimulation of the effector phospholipase C beta. The binding site for G alpha(q/ll) resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha(q/ll) binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G alpha(q/ll). These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G alpha(q/ll) binding defects impair recruitment to the plasma membrane by activated G alpha(q) and regulation of G alpha(q)-stimulated phospholipase C beta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G alpha binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8. The adenomatous polyposis coli (APC) binding site of axin involves residues on alpha helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G alpha(q/ll) binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha 5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.