RESUMO
G protein-coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes-namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)-based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of ß-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Endocitose/fisiologia , Endossomos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Células HEK293 , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologia , Transdução de Sinais/fisiologia , beta-Arrestinas/fisiologiaRESUMO
ß-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that ß-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that ß-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of ß-arrestin revealed that endogenous ß-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of â¼90 s, 100 times longer than for unactivated receptors. PAR2/ß-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, ß-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Células HEK293 , Humanos , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfatidilinositóis/metabolismo , Fosforilação , Ligação Proteica , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , beta-Arrestina 1/metabolismo , beta-Arrestinas/fisiologiaRESUMO
Asthma is a highly prevalent disorder characterized by chronic lung inflammation and reversible airways obstruction. Pathophysiological features of asthma include episodic and reversible airway narrowing due to increased bronchial smooth muscle shortening in response to external and host-derived mediators, excessive mucus secretion into the airway lumen, and airway remodeling. The aberrant airway smooth muscle (ASM) phenotype observed in asthma manifests as increased sensitivity to contractile mediators (EC50) and an increase in the magnitude of contraction (Emax); collectively these attributes have been termed "airways hyper-responsiveness" (AHR). This defining feature of asthma can be promoted by environmental factors including airborne allergens, viruses, and air pollution and other irritants. AHR reduces airway caliber and obstructs airflow, evoking clinical symptoms such as cough, wheezing and shortness of breath. G-protein-coupled receptors (GPCRs) have a central function in asthma through their impact on ASM and airway inflammation. Many but not all treatments for asthma target GPCRs mediating ASM contraction or relaxation. Here we discuss the roles of specific GPCRs, G proteins, and their associated signaling pathways, in asthma, with an emphasis on endogenous mechanisms of GPCR regulation of ASM tone and lung inflammation including regulators of G-protein signaling (RGS) proteins, G-protein coupled receptor kinases (GRKs), and ß-arrestin.
Assuntos
Asma , Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Transdução de Sinais , Asma/metabolismo , Quinases de Receptores Acoplados a Proteína G/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Humanos , Proteínas RGS/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , beta-Arrestinas/fisiologiaRESUMO
Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch. MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeine alone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a ß-arrestin-1âdependent manner. Codeine-triggered ß-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions.
Assuntos
Codeína/farmacologia , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de IgE/fisiologia , Receptores de Neuropeptídeos/fisiologia , Receptores Opioides/fisiologia , Pele/efeitos dos fármacos , beta-Arrestinas/fisiologia , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Mastócitos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Neoplastically transformed astrocytes express functionally active cell surface ß adrenergic receptors (ßARs). Treatment of glioma models in vitro and in vivo with ß adrenergic agonists variably amplifies or attenuates cellular proliferation. In the majority of in vivo models, ß adrenergic agonists generally reduce cellular proliferation. However, treatment with ß adrenergic agonists consistently reduces tumor cell invasive potential, angiogenesis, and metastasis. ß adrenergic agonists induced decreases of invasive potential are chiefly mediated through reductions in the expression of matrix metalloproteinases types 2 and 9. Treatment with ß adrenergic agonists also clearly reduce tumoral neoangiogenesis, which may represent a putatively useful mechanism to adjuvantly amplify the effects of bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor receptor. We may accordingly designate ßagonists to represent an enhancer of bevacizumab. The antiangiogenic effects of ß adrenergic agonists may thus effectively render an otherwise borderline effective therapy to generate significant enhancement in clinical outcomes. ß adrenergic agonists upregulate expression of the major histocompatibility class II DR alpha gene, effectively potentiating the immunogenicity of tumor cells to tumor surveillance mechanisms. Authors have also demonstrated crossmodal modulation of signaling events downstream from the ß adrenergic cell surface receptor and microtubular polymerization and depolymerization. Complex effects and desensitization mechanisms of the ß adrenergic signaling may putatively represent promising therapeutic targets. Constant stimulation of the ß adrenergic receptor induces its phosphorylation by ß adrenergic receptor kinase (ßARK), rendering it a suitable substrate for alternate binding by ß arrestins 1 or 2. The binding of a ß arrestin to ßARK phosphorylated ßAR promotes receptor mediated internalization and downregulation of cell surface receptor and contemporaneously generates a cell surface scaffold at the ßAR. The scaffold mediated activation of extracellular regulated kinase 1/2, compared with protein kinase A mediated activation, preferentially favors cytosolic retention of ERK1/2 and blunting of nuclear translocation and ensuant pro-transcriptional activity. Thus, ßAR desensitization and consequent scaffold assembly effectively retains the cytosolic homeostatic functions of ERK1/2 while inhibiting its pro-proliferative effects. We suggest these mechanisms specifically will prove quite promising in developing primary and adjuvant therapies mitigating glioma growth, angiogenesis, invasive potential, and angiogenesis. We suggest generating compounds and targeted mutations of the ß adrenergic receptor favoring ß arrestin binding and scaffold facilitated activation of ERK1/2 may hold potential promise and therapeutic benefit in adjuvantly treating most or all cancers. We hope our discussion will generate fruitful research endeavors seeking to exploit these mechanisms.
Assuntos
Glioma/metabolismo , Receptores Adrenérgicos beta/metabolismo , beta-Arrestinas/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Animais , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células , Glioma/genética , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Transporte Proteico , Receptores Adrenérgicos beta 2/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Coluna Vertebral/genética , Neoplasias da Coluna Vertebral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases de Receptores Adrenérgicos beta/metabolismo , beta-Arrestinas/fisiologiaRESUMO
In goldfish, two native isoforms of gonadotropin-releasing hormone (GnRH2 and GnRH3) stimulate luteinizing hormone (LH) and growth hormone (GH) release from pituitary cells through activation of cell-surface GnRH-receptors (GnRHRs) on gonadotrophs and somatotrophs. Interestingly, GnRH2 and GnRH3 induce LH and GH release via non-identical post-receptor signal transduction pathways in a ligand- and cell-type-selective manner. In this study, we examined the involvement of ß-arrestins in the control of GnRH-induced LH and GH secretion from dispersed goldfish pituitary cells. Treatment with Barbadin, which interferes with ß-arrestin and ß2-adaptin subunit interaction, reduced LH responses to GnRH2 and GnRH3, as well as GH responses to GnRH2; but enhanced GnRH3-induced GH secretion. Barbadin also had positive influences on basal hormone release, and basal GH release in particular, as well as basal activity of extracellular signal-regulated kinase (ERK) and GnRH-induced ERK activation. These findings indicate that ß-arrestins play permissive roles in the control of GnRH-stimulated LH release. However, in somatotrophs, ß-arrestins, perhaps by mediating agonist-selective endosomal trafficking of engaged GnRHRs, participate in GnRH-isoform-specific GH release responses (stimulatory and inhibitory for GnRH2-GnRHR and GnRH3-GnRHR activation, respectively). The correlative stimulatory influences of Barbadin on basal hormone release and ERK activation suggest that ß-arrestins may negatively regulate basal secretion through modulation of basal ERK activity. These results provide the first direct evidence of a role for ß-arrestins in hormone secretion from an untransformed primary pituitary cell model, and establish these proteins as important receptor-proximal players in mediating functional selectivity downstream of goldfish GnRHRs.
Assuntos
Carpa Dourada , Gonadotrofos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Somatotrofos/efeitos dos fármacos , beta-Arrestinas/fisiologia , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Carpa Dourada/metabolismo , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio do Crescimento/metabolismo , Hormônio Luteinizante/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Somatotrofos/metabolismo , beta-Arrestinas/antagonistas & inibidoresRESUMO
Bitter taste receptor-14 (TAS2R14) is a GPCR also expressed on human airway smooth muscle cells, which signals to intracellular [Ca2+], resulting in relaxation of the airway, and is a novel target for bronchodilators. Here, we examine long-term, agonist-promoted down-regulation of TAS2R14 expression because tachyphylaxis would be an undesirable therapeutic characteristic. Five TAS2R structurally distinct full agonists were studied to ascertain biasing away from down-regulation. Agonist exposure for 18 h caused minimal desensitization by diphenhydramine (DPD) compared with â¼50% desensitization with all other agonists. Agonists evoked ß-arrestin recruitment to TAS2R14, which was not seen with a phosphoacceptor-deficient mutant, TAS2R14-10A. All agonists except for DPD also caused subsequent TAS2R14 internalization and trafficking via early and late endosomes to down-regulation. TAS2R14-10A failed to undergo these events with any agonist. Molecular docking showed that DPD has specific interactions deep within a binding pocket that are not observed with the other agonists, which may lock the receptor in a conformation that does not internalize and therefore does not undergo down-regulation. Thus, TAS2R14 is subject to ß-arrestin-mediated internalization and subsequent down-regulation with chronic exposure to most agonists. However, by manipulating the agonist structure, biasing toward G-protein coupling but away from long-term down-regulation can be achieved.-Woo, J. A., Castaño, M., Goss, A., Kim, D., Lewandowski, E. M., Chen, Y., Liggett, S. B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologia , Cálcio/metabolismo , Difenidramina/farmacologia , Endossomos/fisiologia , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , beta-Arrestinas/fisiologiaRESUMO
Our previous study demonstrated that remifentanil, an opioid agonist, conferred profound liver protection during hepatic ischemia reperfusion injury (HIRI), in which Toll-like receptors (TLRs) played a crucial role in mediating the inflammatory responses. ß-arrestin2, a well-known mu opioid receptor desensitizer, is also a negatively regulator of Toll-like receptor 4 (TLR4)-mediated inflammatory reactions in a mitogen-activated protein kinase (MAPK)-dependent manner. Using the rodent models of hepatic ischemia reperfusion injury both in wild type and TLR4 knockout (TLR4 KO) mice, we found that remifentanil preconditioning could inhibit the expression of TLR4 and reduce the inflammatory response induced by HIRI in wild type but not in TLR4 KO mice. For the in-vitro study, LPS was used to treat RAW264.7 macrophage cells to mimic the inflammatory response induced by HIRI. Remifentanil increased ß-arrestin2 expression both in vivo and in vitro, while after silencing ß-arrestin2 RNA, the effect of remifentanil in reducing cell death and apoptosis, as well as decreasing phosphorylation of ERK and JNK were abolished in RAW264.7 cells. These data suggested that remifentanil could ameliorate mice HIRI through upregulating ß-arrestin2 expression, which may function as a key molecule in bridging opioid receptor and TLR4 pathway.