RESUMO
The heterotrimeric G protein family plays essential roles during a varied array of cellular events; thus, its deregulation can seriously alter signaling events and the overall state of the cell. Heterotrimeric G-proteins have three subunits (α, ß, γ) and are subdivided into four families, Gαi, Gα12/13, Gαq, and Gαs. These proteins cycle between an inactive Gα-GDP state and active Gα-GTP state, triggered canonically by the G-protein coupled receptor (GPCR) and by other accessory proteins receptors independent also known as AGS (Activators of G-protein Signaling). In this review, we summarize research data specific for the Gαi family. This family has the largest number of individual members, including Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, and Gαz, and constitutes the majority of G proteins α subunits expressed in a tissue or cell. Gαi was initially described by its inhibitory function on adenylyl cyclase activity, decreasing cAMP levels. Interestingly, today Gi family G-protein have been reported to be importantly involved in the immune system function. Here, we discuss the impact of Gαi on non-canonical effector proteins, such as c-Src, ERK1/2, phospholipase-C (PLC), and proteins from the Rho GTPase family members, all of them essential signaling pathways regulating a wide range of physiological processes.
RESUMO
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
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
Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.
Assuntos
Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Crista Neural/citologia , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Adesões Focais/efeitos dos fármacos , Modelos Biológicos , Morfolinos/farmacologia , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Xenopus/embriologia , Quinases da Família src/metabolismoRESUMO
The neural crest (NC) is a transient embryonic cell population that migrates extensively during development. Ric-8A, a guanine nucleotide exchange factor (GEF) for different Gα subunits regulates cranial NC (CNC) cell migration in Xenopus through a mechanism that still remains to be elucidated. To properly migrate, CNC cells establish an axis of polarization and undergo morphological changes to generate protrusions at the leading edge and retraction of the cell rear. Here, we aim to study the role of Ric-8A in cell polarity during CNC cell migration by examining whether its signaling affects the localization of GTPase activity in Xenopus CNC using GTPase-based probes in live cells and aPKC and Par3 as polarity markers. We show that the levels of Ric-8A are critical during migration and affect the localization of polarity markers and the subcellular localization of GTPase activity, suggesting that Ric-8A, probably through heterotrimeric G-protein signaling, regulates cell polarity during CNC migration.
Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Crista Neural/metabolismo , Crista Neural/fisiologia , Animais , Transdução de Sinais/fisiologia , XenopusRESUMO
Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gßγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons.
Assuntos
Espinhas Dendríticas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt-5a/metabolismo , Animais , Linhagem Celular Transformada , Espinhas Dendríticas/genética , Receptores Frizzled , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Hipocampo/citologia , Camundongos , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/genética , Proteína Wnt-5a/genéticaRESUMO
In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gßγ signaling, ßARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, ßARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and ß-myosin heavy chain (ß-MHC), was prevented by AG538, PTX, ßARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/ßγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin.