RESUMO
Diet and the gut microbiota have a profound influence on physiology and health, however, mechanisms are still emerging. Here we outline several pathways that gut microbiota products, particularly short-chain fatty acids (SCFAs), use to maintain gut and immune homeostasis. Dietary fibre is fermented by the gut microbiota in the colon, and large quantities of SCFAs such as acetate, propionate, and butyrate are produced. Dietary fibre and SCFAs enhance epithelial integrity and thereby limit systemic endotoxemia. Moreover, SCFAs inhibit histone deacetylases (HDAC), and thereby affect gene transcription. SCFAs also bind to 'metabolite-sensing' G-protein coupled receptors (GPCRs) such as GPR43, which promotes immune homeostasis. The enormous amounts of SCFAs produced in the colon are sufficient to lower pH, which affects the function of proton sensors such as GPR65 expressed on the gut epithelium and immune cells. GPR65 is an anti-inflammatory Gαs-coupled receptor, which leads to the inhibition of inflammatory cytokines. The importance of GPR65 in inflammatory diseases is underscored by genetics associated with the missense variant I231L (rs3742704), which is associated with human inflammatory bowel disease, atopic dermatitis, and asthma. There is enormous scope to manipulate these pathways using specialized diets that release very high amounts of specific SCFAs in the gut, and we believe that therapies that rely on chemically modified foods is a promising approach. Such an approach includes high SCFA-producing diets, which we have shown to decrease numerous inflammatory western diseases in mouse models. These diets operate at many levels - increased gut integrity, changes to the gut microbiome, and promotion of immune homeostasis, which represents a new and highly promising way to prevent or treat human disease.
Assuntos
Acetatos , Ácidos Graxos Voláteis , Animais , Camundongos , Humanos , Ácidos Graxos Voláteis/metabolismo , Butiratos/metabolismo , Fibras na Dieta , ImunomodulaçãoRESUMO
The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here, we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalizes and localizes to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulate receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 is still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalize and localize to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.
Assuntos
Endossomos , Prótons , Animais , Membrana Celular , Transdução de Sinais , MamíferosRESUMO
Changes in extracellular proton concentrations occur in a variety of tissues over a range of timescales under physiological conditions and also accompany virtually all pathologies, notably cancers, stroke, inflammation and trauma. Proton-activated, G protein coupled receptors are already partially active at physiological extracellular proton concentrations and their activity increases with rising proton concentrations. Their ability to monitor and report changes in extracellular proton concentrations and hence extracellular pH appears to be involved in a variety of processes, and it is likely to mirror and in some cases promote disease progression. Unsurprisingly, therefore, these pH-sensing receptors (pHR) receive increasing attention from researchers working in an expanding range of research areas, from cellular neurophysiology to systemic inflammatory processes. This review is looking at progress made in the field of pHRs over the past few years and also highlights outstanding issues.
Assuntos
Neoplasias , Prótons , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Concentração de Íons de Hidrogênio , InflamaçãoRESUMO
Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H+). How these receptors have uniquely obtained this ability is unknown. Here, we show these receptors evolved the capability to sense H+ signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed deep variant profiling, a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using deep variant profiling and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. In addition, we found that an allosteric modulator of many class A GPCRs, Na+, synergistically regulated pH sensing by maintaining the pKa values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H+ and Na+. Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing.
Assuntos
Prótons , Receptores Acoplados a Proteínas G/metabolismo , Sódio/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Sítios de Ligação , Cátions Monovalentes , Biologia Computacional/métodos , Evolução Molecular , Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutação , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sódio/químicaRESUMO
PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation. The extracellular proton-sensing mechanisms are inherent in various cells including airway structural cells, although their physiological and pathophysiological roles in bronchial smooth muscles (BSMs) are not fully understood. In the present study, to explore the functional role of extracellular acidification on the BSM contraction, the isolated mouse BSMs were exposed to acidic pH under contractile stimulation. METHODS AND RESULTS: The RT-PCR analyses revealed that the proton-sensing G protein-coupled receptors were expressed both in mouse BSMs and cultured human BSM cells. In the mouse BSMs, change in the extracellular pH from 8.0 to 6.8 caused an augmentation of contraction induced by acetylcholine. Interestingly, the acidic pH-induced BSM hyper-contraction was further augmented in the mice that were sensitized and repeatedly challenged with ovalbumin antigen. In this animal model of asthma, upregulations of G protein-coupled receptor 68 (GPR68) and GPR65, that were believed to be coupled with Gq and Gs proteins respectively, were observed, indicating that the acidic pH could cause hyper-contraction probably via an activation of GPR68. However, psychosine, a putative antagonist for GPR68, failed to block the acidic pH-induced responses. CONCLUSION: These findings suggest that extracellular acidification contributes to the airway hyperresponsiveness, a characteristic feature of bronchial asthma. Further studies are required to identify the receptor(s) responsible for sensing extracellular protons in BSM cells.
Assuntos
Asma , Hiper-Reatividade Brônquica , Acetilcolina/efeitos adversos , Acetilcolina/metabolismo , Animais , Brônquios , Hiper-Reatividade Brônquica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/metabolismo , Ovalbumina , Prótons , Psicosina/efeitos adversos , Psicosina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Efficient synthetic approaches for the incorporation of nitrogen into polyaromatic compounds (PACs) in different patterns as stabilising moiety for π-extended systems and modification tool for optoelectronic properties remain a challenge until today. Herein, we developed a new versatile pathway to napthyridine-based PACs as non-symmetric and regioisomeric pendant to pyrazine-based PACs. A combination of a gold-catalysed synthesis of 2-aminoquinolines and the development of an in situ desulfonation and condensation of these precursors are the key steps of the protocol. The shape and type of attached functional groups of the PACs can be designed in a late stage of the overall synthetic procedure by the chosen anthranile and backbone of the ynamide introduced in the gold-catalysed step. Single-crystal X-ray diffraction and the investigation of electronic properties of the compounds show the influence of the attached substituents. All naphthyridine-based PACs show halochromic behaviour implying their use as highly sensitive proton sensor in non-protic solvents.
RESUMO
Acid-sensing ion channels (ASICs) are proton-gated ion channels broadly expressed in the vertebrate nervous system, converting decreased extracellular pH into excitatory sodium current. ASICs were previously thought to be a vertebrate-specific branch of the DEG/ENaC family, a broadly conserved but functionally diverse family of channels. Here, we provide phylogenetic and experimental evidence that ASICs are conserved throughout deuterostome animals, showing that ASICs evolved over 600 million years ago. We also provide evidence of ASIC expression in the central nervous system of the tunicate, Oikopleura dioica Furthermore, by comparing broadly related ASICs, we identify key molecular determinants of proton sensitivity and establish that proton sensitivity of the ASIC4 isoform was lost in the mammalian lineage. Taken together, these results suggest that contributions of ASICs to neuronal function may also be conserved broadly in numerous animal phyla.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Cordados/fisiologia , Animais , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Camundongos , Filogenia , Isoformas de ProteínasRESUMO
Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis. In the kidney, OGR1 mRNA was found in cells of the glomerulus, proximal tubule, and interstitium including endothelial cells. Wild type (OGR1+/+) and OGR1 knockout (OGR1-/-) mice were given standard chow without (control) or loaded with ammonium chloride for one or seven days to induce acute or chronic metabolic acidosis, respectively. No differences in responding to the acid load were observed in the knockout mice, except for higher plasma bicarbonate after one day. Bone mineral density, resorption activity of osteoclasts, and urinary deoxypyridinoline were similar between genotypes. During metabolic acidosis the expression levels of key proteins involved in calcium reabsorption, i.e. the sodium/proton exchanger (NHE3), the epithelial calcium-selective channel TRPV5, and the vitamin D-dependent calcium binding protein calbindin-D28k were all higher in the knockout mice compared to wild type mice. This is consistent with the previous demonstration that OGR1 reduces NHE3 activity in proximal tubules of mice. Wild-type mice displayed a non-linear positive association between urinary proton and calcium excretion which was lost in the knockout mice. Thus, OGR1 is a pH sensor involved in the hypercalciuria of metabolic acidosis by controlling NHE3 activity in the proximal tubule. Hence, novel drugs modulating OGR1 activity may improve renal calcium handling.
Assuntos
Acidose , Cálcio , Receptores Acoplados a Proteínas G , Acidose/genética , Animais , Cálcio/metabolismo , Células Endoteliais/metabolismo , Proteínas de Ligação ao GTP , Túbulos Renais Proximais/metabolismo , Camundongos , Camundongos Knockout , Prótons , Receptores Acoplados a Proteínas G/genética , Trocador 3 de Sódio-HidrogênioRESUMO
Acid-base homeostasis is essential for life. The macromolecules upon which living organisms depend are sensitive to pH changes, and physiological systems use the equilibrium between carbon dioxide, bicarbonate, and protons to buffer their pH. Biological processes and environmental insults are constantly challenging an organism's pH; therefore, to maintain a consistent and proper pH, organisms need sensors that measure pH and that elicit appropriate responses. Mammals use multiple sensors for measuring both intracellular and extracellular pH, and although some mammalian pH sensors directly measure protons, it has recently become apparent that many pH-sensing systems measure pH via bicarbonate-sensing soluble adenylyl cyclase.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Equilíbrio Ácido-Base/fisiologia , Desequilíbrio Ácido-Base/fisiopatologia , Homeostase/fisiologia , Adenilil Ciclases , Animais , Bicarbonatos , Dióxido de Carbono , Humanos , Concentração de Íons de Hidrogênio , PrótonsRESUMO
GPR68 (or ovarian cancer G protein-coupled receptor 1, OGR1) is a proton-sensing G-protein-coupled receptor (GPCR) that responds to extracellular acidity and regulates a variety of cellular functions. Acidosis is considered a defining hallmark of the tumor microenvironment (TME). GPR68 expression is highly upregulated in numerous types of cancer. Emerging evidence has revealed that GPR68 may play crucial roles in tumor biology, including tumorigenesis, tumor growth, and metastasis. This review summarizes current knowledge regarding GPR68-its expression, regulation, signaling pathways, physiological roles, and functions it regulates in human cancers (including prostate, colon and pancreatic cancer, melanoma, medulloblastoma, and myelodysplastic syndrome). The findings provide evidence for GPR68 as a potentially novel therapeutic target but in addition, we note challenges in developing drugs that target GPR68.
Assuntos
Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Humanos , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Rheumatoid arthritis (RA), characterized by chronic inflammation of synovial joints, is often associated with ongoing pain and increased pain sensitivity. Chronic pain that comes with RA turns independent, essentially becoming its own disease. It could partly explain that a significant number (50%) of RA patients fail to respond to current RA therapies that focus mainly on suppression of joint inflammation. The acute phase of pain seems to associate with joint inflammation in early RA. In established RA, the chronic phase of pain could be linked to inflammatory components of neuron-immune interactions and noninflammatory components. Accumulating evidence suggests that the initial inflammation and autoimmunity in RA (preclinical RA) begin outside of the joint and may originate at mucosal sites and alterations in the composition of microbiota located at mucosal sites could be essential for mucosal inflammation, triggering joint inflammation. Fibroblast-like synoviocytes in the inflamed joint respond to cytokines to release acidic components, lowering pH in synovial fluid. Extracellular proton binds to proton-sensing ion channels, and G-protein-coupled receptors in joint nociceptive fibers may contribute to sensory transduction and release of neurotransmitters, leading to pain and hyperalgesia. Activation of peripheral sensory neurons or nociceptors further modulates inflammation, resulting in neuroinflammation or neurogenic inflammation. Peripheral and central nerves work with non-neuronal cells (such as immune cells, glial cells) in concert to contribute to the chronic phase of RA-associated pain. This review will discuss actions of proton-sensing receptors on neurons or non-neuronal cells that modulate RA pathology and associated chronic pain, and it will be beneficial for the development of future therapeutic treatments.
Assuntos
Artrite Reumatoide/fisiopatologia , Canais Iônicos/fisiologia , Nociceptores/fisiologia , Dor/fisiopatologia , Receptores Acoplados a Proteínas G/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Hiperalgesia/fisiopatologia , Prótons , Líquido Sinovial/químicaRESUMO
KEY POINTS: The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells. OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues. Activation of TRPC4-containing channels in DAOY cells, but not non-transformed granule precursor cells, results in prominent increases in [Ca2+ ]i and promotes cell motility in wound healing and transwell migration assays. Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca2+ ]i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4. Our results suggest that OGR1 enhances expression of TRPC4-containing channels that contribute to enhanced invasion and metastasis of granule precursor-derived human medulloblastoma. ABSTRACT: Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton-sensing Gq -coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton-potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed TRPC4. In DAOY cells, activation of TRPC4-containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration were observed. Our results suggest that OGR1-dependent increases in TRPC4 expression may favour formation of highly Ca2+ -permeable TRPC4-containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB.
Assuntos
Cálcio/metabolismo , Meduloblastoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Cerebelo/citologia , Humanos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Permeabilidade , Canais de Cátion TRPC/genéticaRESUMO
Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.
Assuntos
Receptores Acoplados a Proteínas G/genética , Proteínas de Peixe-Zebra/genética , Animais , Cobalto/farmacologia , AMP Cíclico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Manganês/farmacologia , Regiões Promotoras Genéticas/genética , Prótons , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/genéticaRESUMO
Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Concentração de Íons de Hidrogênio , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Proton-sensing G-protein-coupled receptors (GPCRs; OGR1, GPR4, G2A, TDAG8), with full activation at pH 6.4 â¼ 6.8, are important to pH homeostasis, immune responses and acid-induced pain. Although G2A mediates the G13-Rho pathway in response to acid, whether G2A activates Gs, Gi or Gq proteins remains debated. In this study, we examined the response of this fluorescence protein-tagged OGR1 family to acid stimulation in HEK293T cells. G2A did not generate detectable intracellular calcium or cAMP signals or show apparent receptor redistribution with moderate acid (pH ≥ 6.0) stimulation but reduced cAMP accumulation under strong acid stimulation (pH ≤ 5.5). Surprisingly, coexpression of OGR1- and G2A-enhanced proton sensitivity and proton-induced calcium signals. This alteration is attributed to oligomerization of OGR1 and G2A. The oligomeric potential locates receptors at a specific site, which leads to enhanced proton-induced calcium signals through channels.
Assuntos
Sinalização do Cálcio/genética , Proteínas de Ciclo Celular/química , Prótons , Receptores Acoplados a Proteínas G/química , Ácidos/química , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , AMP Cíclico/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Multimerização Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors.
Assuntos
Prótons , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Transdução de Sinais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF.
Assuntos
Ácidos/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Concentração de Íons de Hidrogênio , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismoRESUMO
Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Microscopia Confocal , Ovariectomia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.
Assuntos
Simulação de Dinâmica Molecular , Prótons , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Histidina/metabolismo , Histidina/química , Mutagênese Sítio-DirigidaRESUMO
Extracellular acidification of tumors is common. Through proton-sensing ion channels or proton-sensing G protein-coupled receptors (GPCRs), tumor cells sense extracellular acidification to stimulate a variety of intracellular signaling pathways including the calcium signaling, which consequently exerts global impacts on tumor cells. Proton-sensing ion channels, and proton-sensing GPCRs have natural advantages as drug targets of anticancer therapy. However, they and the calcium signaling regulated by them attracted limited attention as potential targets of anticancer drugs. In the present review, we discuss the progress in studies on proton-sensing ion channels, and proton-sensing GPCRs, especially emphasizing the effects of calcium signaling activated by them on the characteristics of tumors, including proliferation, migration, invasion, metastasis, drug resistance, angiogenesis. In addition, we review the drugs targeting proton-sensing channels or GPCRs that are currently in clinical trials, as well as the relevant potential drugs for cancer treatments, and discuss their future prospects. The present review aims to elucidate the important role of proton-sensing ion channels, GPCRs and calcium signaling regulated by them in cancer initiation and development. This review will promote the development of drugs targeting proton-sensing channels or GPCRs for cancer treatments, effectively taking their unique advantage as anti-cancer drug targets.