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Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFß receptor or TGFBR3), a multi-faceted proteoglycan TGFß co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan.
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The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.
Assuntos
Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-akt , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação , Células HEK293 , Humanos , Animais , CamundongosRESUMO
Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF's catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/genética , Células Endoteliais/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismoRESUMO
Cancer cell migration involves a repertoire of signaling proteins that lead cytoskeleton reorganization as a critical step in metastatic dissemination. RhoGEFs are multidomain effectors that integrate signaling inputs to activate the molecular switches that orchestrate actin cytoskeleton reorganization. Ephexins, a group of five RhoGEFs, play oncogenic roles in invasive and metastatic cancer, leading to a mechanistic hypothesis about their function as signaling nodes assembling functional complexes that guide cancer cell migration. To identify clinically significant Ephexin signaling partners, we applied three systematic data mining strategies, based on the screening of essential Ephexins in multiple cancer cell lines and the identification of coexpressed signaling partners in the TCGA cancer patient datasets. Based on the domain architecture of encoded proteins and gene ontology criteria, we selected Ephexin signaling partners with a role in cytoskeletal reorganization and cell migration. We focused on Ephexin3/ARHGEF5, identified as an essential gene in multiple cancer cell types. Based on significant coexpression data and coessentiality, the signaling repertoire that accompanies Ephexin3 corresponded to three groups: pan-cancer, cancer-specific and coessential. To further select the Ephexin3 signaling partners likely to be relevant in clinical settings, we first identified those whose high expression was statistical linked to shorter patient survival. The resulting Ephexin3 transcriptional signatures represent significant accumulated risk, predictive of shorter survival, in 17 cancer types, including PAAD, LUAD, LGG, OSC, AML, KIRC, THYM, BLCA, LIHC and UCEC. The signaling landscape that accompanies Ephexin3 in various cancer types included the tyrosine kinase receptor MET and the tyrosine phosphatase receptor PTPRF, the serine/threonine kinases MARK2 and PAK6, the Rho GTPases RHOD, RHOF and RAC1, and the cytoskeletal regulator DIAHP1. Our findings set the basis to further explore the role of Ephexin3/ARHGEF5 as an essential effector and signaling hub in cancer cell migration.
Assuntos
Neoplasias , Microambiente Tumoral , Humanos , Prognóstico , Transdução de Sinais , Movimento Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
Calcium sensing receptor (CaSR), a class C GPCR, regulates essential secretory pathways, involving communication between endocytic and secretory Rab GTPases, via still to be fully defined molecular mechanisms. To address how communication between endocytic and secretory vesicles occurs, we hypothesized that CaSR activates endocytic Rab11A-dependent effector pathways acting upstream of Rab27B-regulated secretion. We found that Rab11A is critical to promote Rab27B-dependent secretion of chemotactic and inflammatory factors, including IL-8, CCL2/MCP-1, and IL1-ß, in response to CaSR stimulation. It also attenuates secretion of IL-6. The process is mediated by endosomal PI3-kinases, Vps34 and PI3KC2α, which promote Rab27B activation. Rab11A interacts with and activates MADD, a guanine exchange factor for Rab3, and Rab27A/B. Mechanistically, CaSR drives Rab11A-dependent coupling of recycling endosomes to secretory-vesicles via endosomal PI3K-mediated activation of a MADD/Rab27B pathway.
RESUMO
LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores de Ácidos Lisofosfatídicos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Endossomos/metabolismo , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismoRESUMO
Systematic analysis of tumor transcriptomes, combined with deep genome sequencing and detailed clinical assessment of hundreds of patients, constitutes a powerful strategy aimed to identify potential biomarkers and therapeutic targets to guide personalized treatments. Oncogenic signaling cascades are integrated by multidomain effector proteins such as P-Rex1, a guanine nucleotide exchange factor for the Rac GTPase (RacGEF), known to promote metastatic dissemination of cancer cells. We hypothesized that patients with high P-Rex1 expression and reduced survival might be characterized by a particular set of signaling proteins co-expressed with this effector of cell migration as a central component of a putative signaling hub indicative of poor prognosis. High P-Rex1 expression correlated with reduced survival of TCGA Lower Grade Glioma (LGG) patients. Thus, guided by PREX1 expression, we searched for signaling partners of this RacGEF by applying a systematic unbiased in silico data mining strategy. We identified 30 putative signaling partners that also correlated with reduced patient survival. These included GPCRs such as CXCR3, GPR82, FZD6, as well as MAP3K1, MAP2K3, NEK8, DYRK3 and RPS6KA3 kinases, and PTPN2 and PTPN22 phosphatases, among other transcripts of signaling proteins and phospho-substrates. This PREX1 signaling hub signature correlated with increased risk of shorter survival of LGG patients from independent datasets and coincided with immune and endothelial transcriptomic signatures, indicating that myeloid infiltration and tumor angiogenesis might contribute to worsen brain tumor pathology. In conclusion, P-Rex1 and its putative signaling partners in LGG are indicative of a signaling landscape of the tumor microenvironment that correlates with poor prognosis and might guide the characterization of signaling targets leading the eventual development of immunotherapeutic strategies.
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The G protein-coupled receptors (GPCRs) are plasma membrane proteins that function as sensors of changes in the internal and external milieux and play essential roles in health and disease. They are targets of hormones, neurotransmitters, local hormones (autacoids), and a large proportion of the drugs currently used as therapeutics and for "recreational" purposes. Understanding how these receptors signal and are regulated is fundamental for progress in areas such as physiology and pharmacology. This review will focus on what is currently known about their structure, the molecular events that trigger their signaling, and their trafficking to endosomal compartments. GPCR phosphorylation and its role in desensitization (signaling switching) are also discussed. It should be mentioned that the volume of information available is enormous given the large number and variety of GPCRs. However, knowledge is fragmentary even for the most studied receptors, such as the adrenergic receptors. Therefore, we attempt to present a panoramic view of the field, conscious of the risks and limitations (such as oversimplifications and incorrect generalizations). We hope this will provoke further research in the area. It is currently accepted that GPCR internalization plays a role signaling events. Therefore, the processes that allow them to internalize and recycle back to the plasma membrane are briefly reviewed. The functions of cytoskeletal elements (mainly actin filaments and microtubules), the molecular motors implicated in receptor trafficking (myosin, kinesin, and dynein), and the GTPases involved in GPCR internalization (dynamin) and endosomal sorting (Rab proteins), are discussed. The critical role phosphoinositide metabolism plays in regulating these events is also depicted.
Assuntos
Endossomos , Receptores Acoplados a Proteínas G , Endossomos/metabolismo , Hormônios/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA-MB-231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gßγ-PI3K-mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.
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The G protein-coupled receptors form the most abundant family of membrane proteins and are crucial physiologic players in the homeostatic equilibrium, which we define as health. They also participate in the pathogenesis of many diseases and are frequent targets of therapeutic intervention. Considering their importance, it is not surprising that different mechanisms regulate their function, including desensitization, resensitization, internalization, recycling to the plasma membrane, and degradation. These processes are modulated in a highly coordinated and specific way by protein kinases and phosphatases, ubiquitin ligases, protein adaptors, interaction with multifunctional complexes, molecular motors, phospholipid metabolism, and membrane distribution. This review describes significant advances in the study of the regulation of these receptors by phosphorylation and endosomal traffic (where signaling can take place); we revisited the bar code hypothesis and include two additional observations: 1) that different phosphorylation patterns seem to be associated with internalization and endosome sorting for recycling or degradation, and 2) that, surprisingly, phosphorylation of some G protein-coupled receptors appears to be required for proper receptor insertion into the plasma membrane. SIGNIFICANCE STATEMENT: G protein-coupled receptor phosphorylation is an early event in desensitization/signaling switching, endosomal traffic, and internalization. These events seem crucial for receptor responsiveness, cellular localization, and fate (recycling/degradation) with important pharmacological/therapeutic implications. Phosphorylation sites vary depending on the cells in which they are expressed and on the stimulus that leads to such covalent modification. Surprisingly, evidence suggests that phosphorylation also seems to be required for proper insertion into the plasma membrane for some receptors.
Assuntos
Membrana Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gßγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression-based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein-coupled LPA receptors via the Gßγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gßγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Neoplasias Pulmonares , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Animais , Movimento Celular , Progressão da Doença , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologia , Microambiente TumoralRESUMO
Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1ß, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxia , Proteínas Quinases Dependentes de AMP Cíclico , Citocinas/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosfatidilinositol 3-Quinase , Receptores de Detecção de Cálcio/fisiologia , Via Secretória/fisiologia , Serina-Treonina Quinases TOR , Microambiente Tumoral , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Gßγ marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, Gßγ recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which Gßγ stimulates P-Rex1 has been debated. We hypothesized that Gßγ activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that Gßγ binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with Gßγ, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to Gßγ, swapped into P-Rex1. Gßγ recruited Q-Rhox to the plasma membrane, indicating that Gßγ/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, Gßγ recruits P-Rex1 via the Gßγ/PDZ-PDZ interface followed by a second contact involving the Gßγ/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Células HEK293 , Humanos , Domínios PDZ , Ligação Proteica , Transdução de SinaisRESUMO
Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.
Assuntos
Movimento Celular , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Domínios de Homologia à Plecstrina/genética , Pseudópodes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , FosforilaçãoRESUMO
Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Células HEK293 , Humanos , Insulina/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Isoformas de Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Ésteres de Forbol/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Endossomos/efeitos dos fármacos , Humanos , Proteínas Luminescentes , Metoxamina/farmacologia , Norepinefrina/farmacologia , Oximetazolina/farmacologia , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab5 de Ligação ao GTP/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Proteína Vermelha FluorescenteRESUMO
Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antineoplásicos/química , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/química , Animais , Membrana Celular/metabolismo , Extensões da Superfície Celular/efeitos dos fármacos , Endocitose , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais , Células HEK293 , Humanos , Camundongos , Fosforilação , Transdução de Sinais , Suínos , Proteínas rho de Ligação ao GTP/químicaRESUMO
Cells expressing eGFP-tagged Rab5 (wild-type or the GDP-Rab5 mutant) and the DsRed-tagged α1B-adrenergic receptors were employed and the roles of GRK2 were studied utilizing paroxetine and the dominant-negative mutant of GRK2 (DN-GRK2). The following parameters were studied: a) FRET (as an index of α1B-adrenergic receptor-Rab5 interaction): b) intracellular accumulation of DsRed fluorescence (receptor internalization); c) α1B-adrenergic receptor phosphorylation, and d) noradrenaline-induced increase in intracellular calcium concentration. Noradrenaline increased α1B-adrenergic receptor-Rab5 interaction, which was blocked by paroxetine and by expression of the dominant-negative GRK2 mutant. Similarly, paroxetine and expression of the DN-GRK2 or the GDP-Rab5 mutants markedly decreased receptor internalization, α1B-adrenergic receptor phosphorylation, and attenuated the ability of the adrenergic agonist to induce homologous desensitization (calcium signaling). The S406, 410,412A α1B-adrenergic receptor mutant did not reproduce the actions of GRK2 inhibition. The data indicate that GRK2 and Rab5 play key roles in α1B-adrenergic receptor phosphorylation, internalization, and desensitization. The possibility that Rab5 might form part of a signaling complex is suggested, as well as that GDP-Rab5 might interfere with the ability of GRK2 to catalyze α1B-adrenergic receptor phosphorylation.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Células HEK293 , Humanos , Mutação , Norepinefrina/farmacologia , Paroxetina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Reciprocal communication among cells of the tumor microenvironment contributes to cancer progression. Here, we show that a protumoral population of cultured bone marrow-derived cells (BMDC) containing Tie2+/CD45+/CD11b + cells responded to lung carcinoma cells and reciprocally stimulated them. These cells migrated via heterotrimeric G protein-dependent signaling pathways and strongly activated the PI3K/AKT, ERK and mTOR signaling cascades in response to conditioned media and chemotactic agonists. To get insight into the molecular machinery involved in BMDC migration, we revealed their repertoire of guanine nucleotide exchange factors for Rho GTPases (RhoGEFs) and G proteins in comparison with fresh bone marrow cells, proven that these cell populations had contrasting effects on tumor growth. BMDC exhibited a higher expression of G protein regulated RhoGEFs including P-Rex1, PDZ-RhoGEF, LARG, Trio and some less well characterized RhoGEFs such as ARHGEF5, ARHGEF17 and PLEKHG6. G proteins such as Gα12/13, Gαq, and the small GTPase RhoJ were also highly expressed in BMDC. Our results indicate that Tie2+/CD45+/CD11b + BMDC express a unique variety of chemotactic transducers and effectors potentially linked to their protumoral effect, warranting further studies to their characterization as molecular targets.
RESUMO
Regulatory subunits of protein kinase A (PKA) inhibit its kinase subunits. Intriguingly, their potential as cAMP-dependent signal transducers remains uncharacterized. We recently reported that type I PKA regulatory subunits (RIα) interact with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a chemotactic Rac guanine exchange factor (RacGEF). Because P-REX1 is known to be phosphorylated and inhibited by PKA, its interaction with RIα suggests that PKA regulatory and catalytic subunits may fine-tune P-REX1 activity or those of its target pools. Here, we tested whether RIα acts as a cAMP-dependent factor promoting P-REX1-mediated Rac activation and cell migration. We observed that Gs-coupled EP2 receptors indeed promote endothelial cell migration via RIα-activated P-REX1. Expression of the P-REX1-PDZ1 domain prevented RIα/P-REX1 interaction, P-REX1 activation, and EP2-dependent cell migration, and P-REX1 silencing abrogated RIα-dependent Rac activation. RIα-specific cAMP analogs activated P-REX1, but lost this activity in RIα-knockdown cells, and cAMP pulldown assays revealed that P-REX1 preferentially interacts with free RIα. Moreover, purified RIα directly activated P-REX1 in vitro We also found that the RIα CNB-B domain is critical for the interaction with P-REX1, which was increased in RIα mutants, such as the acrodysostosis-associated mutant, that activate P-REX1 at basal cAMP levels. RIα and Cα PKA subunits targeted distinct P-REX1 molecules, indicated by an absence of phosphorylation in the active fraction of P-REX1. This was in contrast to the inactive fraction in which phosphorylated P-REX1 was present, suggesting co-existence of dual stimulatory and inhibitory effects. We conclude that PKA's regulatory subunits are cAMP-dependent signal transducers.