RESUMEN
Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.
Asunto(s)
Proteínas Portadoras , Ubiquitina-Proteína Ligasas , Unión Proteica , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/metabolismo , Ubiquitina/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismoRESUMEN
We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gß1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gß1. Our work demonstrates a unique relationship between KCTD proteins and Gß1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.
Asunto(s)
Proliferación Celular , Canales de Potasio , Humanos , Proliferación Celular/genética , Sistemas CRISPR-Cas , Edición Génica , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Canales de Potasio/metabolismo , Canales de Potasio/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or ß-arrestins, we showed that Gαq and Gα11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of ß-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or ß-arrestin.
Asunto(s)
Receptor de Angiotensina Tipo 1/química , Angiotensina II/metabolismo , Ingeniería Celular/métodos , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Humanos , Ligandos , Métodos , Conformación Proteica , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with Renilla luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gαq and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, ß-arrestin-biased AT1R agonists could also transmit a Gαq-dependent signal to FP without activation of downstream Gαq signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gαq by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Modelos Moleculares , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de Prostaglandina/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Regulación Alostérica , Transferencia de Energía por Resonancia de Bioluminiscencia , Técnicas Biosensibles , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Células HEK293 , Humanos , Ligandos , Luciferasas de Renilla/química , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C/metabolismo , Multimerización de Proteína , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/genética , Receptores de Oxitocina/agonistas , Receptores de Oxitocina/química , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/química , Receptores de Prostaglandina/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/métodos , Diseño de Fármacos , Colorantes Fluorescentes/síntesis química , Receptores Acoplados a Proteínas G/química , Antagonistas del Receptor de Adenosina A2/síntesis química , Antagonistas del Receptor de Adenosina A2/metabolismo , Secuencia de Aminoácidos , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luciferasas de Renilla/síntesis química , Luciferasas de Renilla/metabolismo , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptor de Adenosina A2A/análisis , Receptor de Adenosina A2A/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and ß-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or ß-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/métodos , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal/fisiología , Angiotensina II/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , LigandosRESUMEN
The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.
Asunto(s)
Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de Prostaglandina/metabolismo , Regulación Alostérica/fisiología , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratas , Transducción de Señal/fisiologíaRESUMEN
Despite the observation of synergistic interactions between the urotensinergic and angiotensinergic systems, the interplay between the urotensin II receptor (hUT) and the angiotensin II type 1 receptor (hAT1R) in regulating cellular signaling remains incompletely understood. Notably, the putative interaction between hUT and hAT1R could engender reciprocal allosteric modulation of their signaling signatures, defining a unique role for these complexes in cardiovascular physiology and pathophysiology. Using a combination of co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and FlAsH BRET-based conformational biosensors, we first demonstrated the physical interaction between hUT and hAT1R. Next, to analyze how this functional interaction regulated proximal and distal hUT- and hAT1R-associated signaling pathways, we used BRET-based signaling biosensors and western blots to profile pathway-specific signaling in HEK 293 cells expressing hUT, hAT1R or both. We observed that hUT-hAT1R heterodimers triggered distinct signaling outcomes compared to their respective parent receptors alone. Notably, co-transfection of hUT and hAT1R has no impact on hUII-induced Gq activation but significantly reduced the potency and efficacy of Ang II to mediate Gq activation. Interestingly, URP, the second hUT endogenous ligand, produce a distinct signaling signature compared to hUII at hUT-hAT1R. Our results therefore suggest that assembly of hUT with hAT1R might be important for allosteric modulation of outcomes associated with specific hardwired signaling complexes in healthy and disease states. Altogether, our work, which potentially explains the interplay observed in native cells and tissues, validates such complexes as potential targets to promote the design of compounds that can modulate heterodimer function selectively.
Asunto(s)
Receptor de Angiotensina Tipo 1 , Urotensinas , Humanos , Angiotensina II , Células HEK293RESUMEN
G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and transmit critical signals from the extracellular to the intracellular spaces. Transcriptomic data of resected breast tumors shows that low mRNA expression of the orphan GPCR GPR52 correlates with reduced overall survival in breast cancer patients, leading to the hypothesis that loss of GPR52 supports breast cancer progression. CRISPR-Cas9 was used to knockout GPR52 in human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line, MCF10A. Loss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells. To determine the in vivo impact of GPR52 loss, MDA-MB-468 cells were injected into zebrafish and loss of GPR52 was associated with a greater total cancer area compared to control cells. RNA-sequencing and proteomic analyses of GPR52-null breast cancer cells reveal an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates production of cAMP, induces some phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some of the GPR52 KO phenotypes. Overall, our results reveal GPR52 loss as a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option in breast cancer.
RESUMEN
The hormone oxytocin (OT) has pleiotropic activities both in the central nervous system as well as in peripheral tissues, including uterotonic effects on the myometrium during parturition. OT effects are mediated by a single transmembrane receptor, belonging to the GPCR (G protein-coupled receptor) superfamily and coupled primarily to Gq- and Gi-containing heterotrimeric G proteins. Upon receptor stimulation, one well-studied downstream effect is activation of the ERK1/2 MAP (mitogen-activated protein) kinase, and studies have shown that induction of COX-2 by OT in the myometrium required ERK1/2 activity. Many studies investigating the role of ERK1/2 in myometrial tissue were based on the use of chemical inhibitors that, to varying degrees, also inhibited ERK5/MAPK7. Here we report that OT activates ERK5 in a human myometrial cell line in a dose- and time-dependent manner through the activation of Gi/o heterotrimers. Using complementary approaches, we demonstrate that OT-induced COX-2 induction and the concomitant release of PGF2α into the media are primarily ERK5-dependent and to a much lesser extent ERK1/2-dependent. Moreover, in contrast to ERK1/2 activation, ERK5 activation is downstream of Gi/o activation. Here, we also found that ERK5 impacted both basal and to a lesser extent, OT-mediated myometrial cell contraction in vitro. Finally, tracking both ERK1/2 and ERK5 activity during different stages of gestation in rat myometrium, we showed that they followed distinct patterns starting at the onset of labor corresponding to the highest COX-2 expression levels. Overall, our results reveal an important, hitherto unrecognized role for ERK5 in myometrial cell contraction involving induction of COX-2. This novel pathway is likely to play an important role in supporting uterine contractions during parturition.
Asunto(s)
Miometrio , Oxitocina , Animales , Femenino , Proteína Quinasa 7 Activada por Mitógenos , Miometrio/metabolismo , Oxitocina/metabolismo , Embarazo , Ratas , Receptores de Oxitocina/metabolismo , Transducción de Señal , Contracción UterinaRESUMEN
BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS: We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS: We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1ß secretion in response to an inflammatory stimulus. CONCLUSIONS: We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.
Asunto(s)
Inflamasomas , Enfermedades Inflamatorias del Intestino , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Interleucina-1beta , Proteína con Dominio Pirina 3 de la Familia NLR , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.
Asunto(s)
Dinoprost/metabolismo , Parto/efectos de los fármacos , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/tratamiento farmacológico , Péptidos/síntesis química , Péptidos/uso terapéutico , Embarazo , Proteína Quinasa C/metabolismoRESUMEN
Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G-protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.
Asunto(s)
Fertilidad , Hormona Luteinizante/metabolismo , Receptores LHRH/química , Receptores LHRH/fisiología , Animales , Pollos , Femenino , Hormona Folículo Estimulante/metabolismo , Ratones , Ratones Transgénicos , Folículo Ovárico/fisiología , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
As the largest family of cell surface receptors, G protein-coupled receptors (GPCRs) represent an important strategic class of therapeutic targets. Attaining a clearer perspective of how such signaling complexes set molecular events in motion could have significant impact on our understanding and treatment of human diseases. As such, many experimental approaches have set out to better understand signaling networks associated with individual receptors to understand signaling architectures and their relationship to signaling outcomes. However, designing in vitro assays aimed at addressing signaling events downstream of single GPCRs must also take into account their propensity to form homo- and heterooligomeric complexes. In the context of GPCR oligomers, physical interactions with a partner protein can have a number of potential consequences, which we will explore in this review. We will also discuss methods used to identify putative dimer partners as well as the various techniques used to study the functional consequences of such complex formation. Since the full functional significance and physiological relevance of GPCR oligomers remains incompletely understood, owing in part to technical limitations, new tools to elucidate molecular mechanisms underlying allosteric co-regulation occurring between two GPCRs are required. Accordingly, using the example of the FP/AT1R heterodimer, we discuss the potential of the FlAsH-BRET approach as a simple tool to reveal how allosteric information is transmitted via conformational rearrangements within putative GPCR complexes and as a means to deorphanize receptors.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Animales , Técnicas Biosensibles , Humanos , Ligandos , Modelos Moleculares , Mutagénesis , Conformación Proteica , Multimerización de Proteína , Receptores de Superficie Celular/químicaRESUMEN
A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different ß-adrenergic ligands to engage five distinct signalling pathways downstream of the ß1-adrenergic receptor (ß1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the ßAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the ß1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Técnicas Biosensibles/métodos , Sistemas CRISPR-Cas , Calcio/metabolismo , Edición Génica , Células HEK293 , Humanos , Ligandos , Fenotipo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Transducción de SeñalRESUMEN
The hypothalamic post-translational processing of oxytocin (OT)-neurophysin precursor involves the formation of C-terminally extended OT forms (OT-X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT-X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor-mediated cardiomyogenic action of OT-Gly-Lys-Arg (OT-GKR). To obtain further insight into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing the OT-GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT-GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT-GKR/EGFP-expressing embryoid bodies and elevated expression of GATA-4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT-GKR on ES cell-derived cardiomyogenesis. Furthermore, patch-clamp experiments demonstrated induction of ventricular phenotypes in OT-GKR/EGFP-transfected and in OT-GKR-treated cardiomyocytes. Increased connexin 43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Corazón/embriología , Miocitos Cardíacos/citología , Oxitocina/metabolismo , Animales , Western Blotting , Células Cultivadas , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional dimers that act as distinct signalling hubs for the integration of cellular signalling. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells (Goupil et al., J Biol Chem 290:3137-3148, 2015; Sleno et al., J Biol Chem 292:12139-12152, 2017). In addition to canonical G protein coupling, GPCRs recruit and engage ß-arrestin-dependent pathways. Using BRET-based biosensors, we demonstrate how to assess recruitment of ß-arrestin-1 and -2 to AT1R and the AT1R/FP dimer in response to Ang II. Surprisingly, ß-arrestin-1 and -2 were recruited to the dimer, in response to PGF2α as well, even though FP alone cannot recruit either ß-arrestin-1 and -2.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Multimerización de Proteína , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Angiotensina II/farmacología , Dinoprost/farmacología , Células HEK293 , Humanos , Multimerización de Proteína/efectos de los fármacos , Receptor de Angiotensina Tipo 1/metabolismoRESUMEN
Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/métodos , Sistemas CRISPR-Cas , Edición Génica/métodos , Luciferasas de Renilla/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Fluorescencia , Humanos , Ligandos , Conformación Proteica , Receptores Acoplados a Proteínas G/genética , Transducción de SeñalRESUMEN
Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2α-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of ß-arrestin recruitment. Using BRET-based biosensors, we assessed the recruitment kinetics of ß-arrestin1/2 to the AT1R/FP dimer, or the parent receptors alone, when stimulated by either Ang II or PGF2α. Using cell lines with CRISPR/Cas9-mediated gene deletion, we also examined the role of G proteins in such recruitment. We observed that Ang II induced a rapid, robust, and sustained recruitment of ß-arrestin1/2 to AT1R and, to a lesser extent, the heterodimer, as expected, since AT1R is a strong recruiter of both ß-arrestin subtypes. However, PGF2α did not induce such recruitment to FP alone, although it did when the AT1R is present as a heterodimer. ß-arrestins were likely recruited to the AT1R partner of the dimer. Gαq, Gα11, Gα12, and Gα13 were all involved to some extent in PGF2α-induced ß-arrestin1/2 recruitment to the dimer as their combined absence abrogated the response, and their separate re-expression was sufficient to partially restore it. Taken together, our data sheds light on a new mechanism whereby PGF2α specifically recruits and signals through ß-arrestin but only in the context of the AT1R/FP dimer, suggesting that this may be a new allosteric signaling entity.