RESUMEN
How G proteins inhibit N-type, voltage-gated, calcium-selective channels (CaV2.2) during presynaptic inhibition is a decades-old question. G proteins Gßγ bind to intracellular CaV2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gßγ acts by selectively inhibiting a subset of the four different CaV2.2 voltage-sensor domains (VSDs I to IV). During regular "willing" gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gßγ, CaV2.2 gating is "reluctant": pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gßγ inhibition of VSDs I and IV underlies reluctant CaV2.2 gating and subsequent presynaptic inhibition.
Asunto(s)
Canales de Calcio Tipo N , Activación del Canal Iónico , Canales de Calcio Tipo N/metabolismo , Animales , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Unión ProteicaRESUMEN
A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.
Asunto(s)
Señalización del Calcio , Calcio , Fosfolipasa C beta , Receptores Acoplados a Proteínas G , Humanos , Fosfolipasa C beta/metabolismo , Fosfolipasa C beta/genética , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Sistemas CRISPR-Cas , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , AMP Cíclico/metabolismo , Animales , Edición Génica , Citosol/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genéticaRESUMEN
AIMS: In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4ß2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. MATERIALS AND METHODS: Loss-of-function strategies and mutants of arrestin2/PKCßII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. KEY FINDINGS: ERK activation via α4ß2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCßII, which exhibited mutual augmentation. Activation of PKCßII by α4ß2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin ß1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCßII signaling cascade, leading to the nuclear entry of PKCßII in a Gßγ-dependent manner. Upon nuclear localization, PKCßII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4ß2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCßII, which is reciprocally facilitated via positive feedback augmentation. SIGNIFICANCE: As α4ß2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4ß2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP , Proteína Quinasa C beta , Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Proteína Quinasa C beta/metabolismo , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Animales , Transducción de Señal , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Núcleo Celular/metabolismoRESUMEN
Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.
Asunto(s)
Adenilil Ciclasas , Proteínas Cullin , Dependencia de Morfina , Animales , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Proteínas Cullin/metabolismo , Ratones , Dependencia de Morfina/metabolismo , Células HEK293 , Humanos , Canales de Potasio/metabolismo , Canales de Potasio/genética , Ratones Endogámicos C57BL , Masculino , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Morfina/farmacología , Ratones Noqueados , Transducción de Señal , AMP Cíclico/metabolismoRESUMEN
Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαßγ proteins, followed by the recruitment of GPCR kinases and ßarrestin (ßarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both ßarr and Gßγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)-ßarr complex scaffolds Gßγ at the plasma membrane through a direct interaction with ßarr, enabling its transport to endosomes. Gßγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of ßarr in mediating sustained G protein signaling.
Asunto(s)
Endosomas , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Transporte de Proteínas , Receptores de Vasopresinas , beta-Arrestinas , Humanos , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/genética , Transducción de SeñalRESUMEN
G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.
Asunto(s)
Quinasas de Receptores Acoplados a Proteína-G , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fosforilación , Animales , Transducción de SeñalRESUMEN
BACKGROUND: Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gß subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling's survival under high salinity remains unclear. RESULTS: We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. CONCLUSIONS: Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Subunidades beta de la Proteína de Unión al GTP , Salinidad , Respuesta de Proteína Desplegada , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Estrés Salino , Regulación de la Expresión Génica de las Plantas , Plantones/genética , Plantones/fisiología , Plantones/metabolismoRESUMEN
Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I , Proteína de Unión al GTP rac1 , Proteínas de Unión al GTP rho , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Microscopía Fluorescente , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/química , Transducción de Señal , Fosfatidilinositol 3-Quinasa Clase I/química , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.
Asunto(s)
Analgésicos Opioides , Tolerancia a Medicamentos , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Ratones Endogámicos C57BL , Morfina , Nocicepción , Transducción de Señal , Animales , Morfina/farmacología , Tolerancia a Medicamentos/fisiología , Transducción de Señal/efectos de los fármacos , Ratones , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Masculino , Analgésicos Opioides/farmacología , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Nocicepción/efectos de los fármacos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/agonistas , Fosfolipasa C beta/metabolismo , XantenosRESUMEN
The heterozygous mutation c.155G > T in GNB2 clinically leads to sinus bradycardia and sinus node dysfunction. Here, patient-specific skin fibroblasts of the mutation carrier were used for Sendai virus reprogramming into human induced-pluripotent stem cells (hiPSC). For generating the isogenic control cell line, a CRISPR/Cas9-mediated HDR-repair of the hiPSCs was carried out. Both generated cell lines (GNB2 SV5528, GNB2 K26) maintained a normal karyotype, cell morphology, pluripotency in immunofluoresence and RT-qPCR analysis. Both hiPSC-lines showed differentiation potential into all three germ layers. Differentiated cardiomyocytes of this isogenic set may pave the way for investigating pharmacological rescue strategies for sinus node dysfunction.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas , Mutación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas CRISPR-Cas/genética , Heterocigoto , Línea Celular , Diferenciación Celular , Síndrome del Seno Enfermo/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismoRESUMEN
Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.
Asunto(s)
Canales de Calcio Tipo T , Subunidades beta de la Proteína de Unión al GTP , Animales , Humanos , Ratas , Canales de Calcio Tipo R , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/química , Proteínas de Transporte de Catión , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Células HEK293 , Cinética , Técnicas de Placa-Clamp , Unión ProteicaRESUMEN
The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gßγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gßγ complexes in the presence of substrates/analogs, revealing two Gßγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gßγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gßγ binding sites and interdomain contacts altered by Gßγ binding suggest that Gßγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gßγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/química , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Sitios de Unión , Pez Cebra , Unión Proteica , Neutrófilos/metabolismo , Modelos Moleculares , Activación Enzimática , Conformación Proteica , Regulación AlostéricaRESUMEN
The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gßγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gßγ and a dimeric form of AC5 that could be involved in its regulation. Gßγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gßγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gßγ and show reduced conditional activation by Gßγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gßγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.
Asunto(s)
Adenilil Ciclasas , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/química , Humanos , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/química , Modelos Moleculares , Células HEK293 , Multimerización de Proteína , Unión Proteica , Animales , Mutación , Conformación ProteicaRESUMEN
Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gß and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gß-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.
Asunto(s)
Resistencia a la Enfermedad , Silenciador del Gen , Glycine max , Enfermedades de las Plantas , Glycine max/genética , Glycine max/inmunología , Glycine max/microbiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad/genética , Comovirus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/inmunología , Regulación de la Expresión Génica de las Plantas , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/inmunología , Xanthomonas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gßγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gßγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.
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Péptido Relacionado con Gen de Calcitonina , Vasos Coronarios , AMP Cíclico , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Vasodilatación , Humanos , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Masculino , Persona de Mediana Edad , AMP Cíclico/metabolismo , Vasodilatación/efectos de los fármacos , Femenino , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Transducción de Señal/efectos de los fármacos , Técnicas In Vitro , Vasodilatadores/farmacologíaRESUMEN
Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.
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Enfermedad de Alzheimer , Subunidades beta de la Proteína de Unión al GTP , Ratones , Humanos , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Estudio de Asociación del Genoma Completo , Ovillos Neurofibrilares/metabolismo , Fenotipo , Genómica , Péptidos beta-Amiloides/genética , Encéfalo/metabolismo , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismoRESUMEN
Heterotrimeric G proteins play key roles in cellular processes. Although phenotypic analyses of Arabidopsis Gß (AGB1) mutants have implicated G proteins in abscisic acid (ABA) signaling, the AGB1-mediated modules involved in ABA responses remain unclear. We found that a partial AGB1 protein was localized to the nucleus where it interacted with ABA-activated VirE2-interacting protein 1 (VIP1) and mitogen-activated protein kinase 3 (MPK3). AGB1 acts as an upstream negative regulator of VIP1 activity by initiating responses to ABA and drought stress, and VIP1 regulates the ABA signaling pathway in an MPK3-dependent manner in Arabidopsis. AGB1 outcompeted VIP1 for interaction with the C-terminus of MPK3, and prevented phosphorylation of VIP1 by MPK3. Importantly, ABA treatment reduced AGB1 expression in the wild type, but increased in vip1 and mpk3 mutants. VIP1 associates with ABA response elements present in the AGB1 promoter, forming a negative feedback regulatory loop. Thus, our study defines a new mechanism for fine-tuning ABA signaling through the interplay between AGB1 and MPK3-VIP1. Furthermore, it suggests a common G protein mechanism to receive and transduce signals from the external environment.
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Proteínas de Arabidopsis , Arabidopsis , Subunidades beta de la Proteína de Unión al GTP , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , FosforilaciónRESUMEN
Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.
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Carcinoma Hepatocelular , Subunidades beta de la Proteína de Unión al GTP , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , alfa-Fetoproteínas/análisis , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Biomarcadores de Tumor/metabolismo , Metilación de ADN , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismoRESUMEN
Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gßγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gßγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gßγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gßγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gßγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.
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Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Insulinas , Ratones , Animales , Calcio/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Exocitosis/fisiología , Proteínas SNARE/genética , Dieta , Obesidad/genética , Adipocitos/metabolismo , Insulinas/metabolismo , Insulina/metabolismoRESUMEN
Heterotrimeric G protein-mediated signaling plays a vital role in physiological and developmental processes in eukaryotes. On the other hand, because of the absence of a G protein-coupled receptor and self-activating mechanism of the Gα subunit, plants appear to have different regulatory mechanisms, which remain to be elucidated, compared to canonical G protein signaling established in animals. Here we report that Arabidopsis heterotrimeric G protein subunits, such as Gα (GPA1) and Gß (AGB1), regulate plant growth under stress conditions through the analysis of heterotrimeric G protein mutants. Flg22-mediated growth inhibition in wild-type roots was found to be caused by a defect in the elongation zone, which was partially blocked in agb1-2 but not gpa1-4. These results suggest that AGB1 may negatively regulate plant growth under biotic stress conditions. In addition, GPA1 and AGB1 exhibited genetically opposite effects on FCA-mediated growth inhibition under heat stress conditions. Therefore, these results suggest that plant G protein signaling is probably related to stress-mediated growth regulation for developmental plasticity in response to biotic and abiotic stress conditions.