RESUMEN
Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Guanosina Trifosfato/química , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neuronas/química , Neuronas/metabolismo , Neuronas/fisiología , Transducción de Señal , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Péptidos/metabolismo , Unión ProteicaRESUMEN
Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Inhibidores de Disociación de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas Nucleares/genética , Ingeniería de Proteínas/métodos , Receptores Acoplados a Proteínas G/genética , Proteínas de Transporte Vesicular/genética , Secuencias de Aminoácidos , Animales , Clonación Molecular , Embrión no Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/metabolismo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/metabolismoRESUMEN
The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in â¼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gßγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the â¼20% of UMs bearing this mutation.
Asunto(s)
Carcinogénesis/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Mutación , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Humanos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Transducción de Señal/genéticaRESUMEN
Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.
Asunto(s)
Secuencia de Consenso , Evolución Molecular , Proteínas de Unión al GTP Heterotriméricas/química , Receptores Acoplados a Proteínas G/química , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans , Humanos , Modelos Moleculares , Conformación Proteica , Transducción de SeñalRESUMEN
Activation of the tyrosine kinase focal adhesion kinase (FAK) upon cell stimulation by the extracellular matrix initiates integrin outside-in signaling. FAK is directly recruited to active integrins, which enhances its kinase activity and triggers downstream signaling like activation of PI3K. We recently described that Gα-interacting, vesicle-associated protein (GIV), a protein up-regulated in metastatic cancers, is also required for outside-in integrin signaling. More specifically, we found that GIV is a non-receptor guanine nucleotide exchange factor that activates trimeric G proteins in response to integrin stimulation to enhance PI3K signaling and tumor cell migration. In contrast, previous reports have established that GIV is involved in phosphotyrosine (Tyr(P))-based signaling in response to growth factor stimulation;i.e.GIV phosphorylation at Tyr-1764 and Tyr-1798 recruits and activates PI3K. Here we show that phosphorylation of GIV at Tyr-1764/Tyr-1798 is also required to enhance PI3K-Akt signaling and tumor cell migration in response to integrin stimulation, indicating that GIV functions in Tyr(P)-dependent integrin signaling. Unexpectedly, we found that activation of FAK, an upstream component of the integrin Tyr(P) signaling cascade, was diminished in GIV-depleted cells, suggesting that GIV is required to establish a positive feedback loop that enhances integrin-FAK signaling. Mechanistically, we demonstrate that this feedback activation of FAK depends on both guanine nucleotide exchange factor and Tyr(P) GIV signaling as well as on their convergence point, PI3K. Taken together, our results provide novel mechanistic insights into how GIV promotes proinvasive cancer cell behavior by working as a signal-amplifying platform at the crossroads of trimeric G protein and Tyr(P) signaling.
Asunto(s)
Integrinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Colágeno/metabolismo , Activación Enzimática , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Neoplasias/patología , FosforilaciónRESUMEN
GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gαi3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células HeLa , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Immunoblotting , Proteínas de Microfilamentos/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA-G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans.
Asunto(s)
Evolución Biológica , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Multimerización de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Evolución Molecular , Proteínas de Unión al GTP/química , Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
There is an increasing number of disease-associated Gα mutations identified from genome-wide sequencing campaigns or targeted efforts. Albright's Hereditary Osteodystrophy (AHO) was the first inherited disease associated with loss-of-function mutations in a G protein (Gαs) and other studies revealed gain-of-function Gα mutations in cancer. Here we attempted to solve the apparent quandary posed by the fact that the same mutation in two different G proteins appeared associated with both AHO and cancer. We first confirmed the presence of an inherited Gαs-R265H mutation from a previously described clinical case report of AHO. This mutation is structurally analogous to Gαo-R243H, an oncogenic mutant with increased activity in vitro and in cells due to rapid nucleotide exchange. We found that, contrary to Gαo-R243H, Gαs-R265H activity is compromised due to greatly impaired nucleotide binding in vitro and in cells. We obtained equivalent results when comparing another AHO mutation in Gαs (D173N) with a counterpart cancer mutation in Gαo (D151N). Gαo-R243H binds nucleotides efficiently under steady-state conditions but releases GDP much faster than the WT protein, suggesting diminished affinity for the nucleotide. These results indicate that the same disease-linked mutation in two different G proteins affects a common biochemical feature (nucleotide affinity) but to a different grade depending on the G protein (mild decrease for Gαo and severe for Gαs). We conclude that Gαs-R265H has dramatically impaired nucleotide affinity leading to the loss-of-function in AHO whereas Gαo-R243H has a mild decrease in nucleotide affinity that causes rapid nucleotide turnover and subsequent hyperactivity in cancer.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Seudohipoparatiroidismo/genética , Seudohipoparatiroidismo/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cromograninas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Linaje , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Adherens junctions (AJs) are a fundamental organizing structure for multicellular life. Although AJs are studied mainly in epithelia, their core function - stabilizing cell contacts by coupling adhesion molecules to the cytoskeleton - is important in diverse tissues. We find that two C. elegans sensory neurons, URX and BAG, require conserved AJ proteins for dendrite morphogenesis. We previously showed that URX and BAG dendrites attach to the embryonic nose via the adhesion molecule SAX-7/L1CAM, acting both in neurons and glia, and then extend by stretch during embryo elongation. Here, we find that a PDZ-binding motif (PB) in the SAX-7 cytoplasmic tail acts with other interaction motifs to promote dendrite extension. Using pull-down assays, we find that the SAX-7 PB binds the multi-PDZ scaffolding protein MAGI-1, which bridges it to the cadherin-catenin complex protein HMP-2/ß-catenin. Using cell-specific rescue and depletion, we find that both MAGI-1 and HMR-1/Cadherin act in glia to non-autonomously promote dendrite extension. Double mutant analysis indicates that each protein can act independently of SAX-7, suggesting a multivalent adhesion complex. The SAX-7 PB motif also binds AFD-1/Afadin, loss of which further enhances sax-7 BAG dendrite defects. As MAGI-1, HMR-1, and AFD-1 are all found in epithelial AJs, we propose that an AJ-like complex in glia promotes dendrite extension.
RESUMEN
The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.
Asunto(s)
Núcleo Celular/metabolismo , Ciclina A/biosíntesis , Ciclina E/biosíntesis , Factor de Transcripción E2F1/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Elementos de Respuesta/fisiología , Transcripción Genética/fisiología , Animales , Núcleo Celular/genética , Proliferación Celular , Ciclina A/genética , Ciclina E/genética , Factor de Transcripción E2F1/genética , Silenciador del Gen , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Estructura Terciaria de ProteínaRESUMEN
The function of IAP has long been limited to an inhibition of apoptosis through their capacity to bind some caspases. Since the expression of these proteins is altered in some tumor samples, IAPs are targets for anticancer therapy and many small molecules have been designed for their capacity to inhibit IAP-caspase interaction. Unexpectedly, these molecules appeared to significantly affect NF-κB activation. In this review, we will discuss the central role of cIAP1, cIAP2 and XIAP in the regulation of NF-κB activating signaling pathways.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/fisiología , FN-kappa B/fisiología , Transducción de Señal/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Animales , Antineoplásicos/farmacología , Proteínas Morfogenéticas Óseas/fisiología , Daño del ADN , Proteínas de Drosophila/fisiología , Humanos , Inmunidad Innata/fisiología , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/genética , Mamíferos , Modelos Genéticos , Terapia Molecular Dirigida , Familia de Multigenes , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/fisiología , Relación Estructura-Actividad , Transcripción Genética , Factor de Crecimiento Transformador beta/fisiología , Proteínas Virales/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Establishment of apicobasal polarity and the organization of the cytoskeleton must operate coordinately to ensure proper epithelial cell shape and function. However, the precise molecular mechanisms by which polarity complexes directly instruct the cytoskeletal machinery to determine cell shape are poorly understood. Here, we define a mechanism by which the PAR polarity complex (PAR3-PAR6-aPKC) at apical cell junctions leads to efficient assembly of the apical actomyosin network to maintain epithelial cell morphology. We found that the PAR polarity complex recruits the protein DAPLE to apical cell junctions, which in turn triggers a two-pronged mechanism that converges upon assembly of apical actomyosin. More specifically, DAPLE directly recruits the actin-stabilizing protein CD2AP to apical junctions and, concomitantly, activates heterotrimeric G protein signaling in a GPCR-independent manner to favor RhoA-myosin activation. These observations establish DAPLE as a direct molecular link between junctional polarity complexes and the formation of apical cytoskeletal assemblies that support epithelial cell shape.
Asunto(s)
Actomiosina , Polaridad Celular , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Microfilamentos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Forma de la Célula , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa C/metabolismoRESUMEN
Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Optogenética/métodos , Proteínas de Plantas , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión , Animales , Avena/genética , Escherichia coli/genética , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genéticaAsunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Transducción de Señal/fisiologíaRESUMEN
Dishevelled-Associating Protein with a high frequency of LEucines (DAPLE) belongs to a group of unconventional activators of heterotrimeric G-proteins that are cytoplasmic factors rather than membrane proteins of the G-protein-coupled receptor superfamily. During neurulation, DAPLE localizes to apical junctions of neuroepithelial cells and promotes apical cell constriction via G-protein activation. While junctional localization of DAPLE is necessary for this function, the factors it associates with at apical junctions or how they contribute to DAPLE-mediated apical constriction are unknown. MPDZ is a multi-PDZ (PSD95/DLG1/ZO-1) domain scaffold present at apical cell junctions whose mutation in humans is linked to nonsyndromic congenital hydrocephalus (NSCH). DAPLE contains a PDZ-binding motif (PBM) and is also mutated in human NSCH, so we investigated the functional relationship between both proteins. DAPLE colocalized with MPDZ at apical cell junctions and bound directly to the PDZ3 domain of MPDZ via its PBM. Much like DAPLE, MPDZ is induced during neurulation in Xenopus and is required for apical constriction of neuroepithelial cells and subsequent neural plate bending. MPDZ depletion also blunted DAPLE--mediated apical constriction of cultured cells. These results show that DAPLE and MPDZ, two factors genetically linked to NSCH, function as cooperative partners at apical junctions and are required for proper tissue remodeling during early stages of neurodevelopment.
Asunto(s)
Polaridad Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Genes Dominantes , Células HEK293 , Humanos , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/química , Neurulación , Dominios PDZ , Unión Proteica , Xenopus laevis/metabolismoRESUMEN
Heterotrimeric G proteins are signaling switches that control organismal morphogenesis across metazoans. In invertebrates, specific GPCRs instruct G proteins to promote collective apical cell constriction in the context of epithelial tissue morphogenesis. In contrast, tissue-specific factors that instruct G proteins during analogous processes in vertebrates are largely unknown. Here, we show that DAPLE, a non-GPCR protein linked to human neurodevelopmental disorders, is expressed specifically in the neural plate of Xenopus laevis embryos to trigger a G protein signaling pathway that promotes apical cell constriction during neurulation. DAPLE localizes to apical cell-cell junctions in the neuroepithelium, where it activates G protein signaling to drive actomyosin-dependent apical constriction and subsequent bending of the neural plate. This function is mediated by a Gα-binding-and-activating (GBA) motif that was acquired by DAPLE in vertebrates during evolution. These findings reveal that regulation of tissue remodeling during vertebrate development can be driven by an unconventional mechanism of heterotrimeric G protein activation that operates in lieu of GPCRs.
Asunto(s)
Embrión no Mamífero/citología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Morfogénesis , Placa Neural/citología , Receptores Acoplados a Proteínas G/metabolismo , Actomiosina/metabolismo , Animales , Células Cultivadas , Constricción , Embrión no Mamífero/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Microfilamentos/genética , Placa Neural/metabolismo , Neurulación , Dominios y Motivos de Interacción de Proteínas , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Xenopus laevis/embriología , Xenopus laevis/fisiología , Pez Cebra/embriología , Pez Cebra/fisiologíaRESUMEN
Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanosina Difosfato/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos/fisiología , Línea Celular , Activación Enzimática/fisiología , Células HEK293 , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/fisiología , Transducción de Señal/fisiologíaRESUMEN
Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein-coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ET(A)R), which is mediated by the G protein (heterotrimeric guanine nucleotide-binding protein) subunit Gα(q/11) and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations inGNAI3, which encodes Gα(i3), but it is unknown whether this G protein has a role within the ET(A)R pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gα(i3) mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gα(i3) mutant proteins that couple to ET(A)R but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gα(q/11) and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.