Proton-sensing G-protein-coupled Receptors Research Articles

Proton-sensing ion channels, GPCRs and calcium signaling regulated by them: implications for cancer.

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.

Protons taken hostage: Dynamic H-bond networks of the pH-sensing GPR68

Proton-sensing G Protein Coupled Receptors (GPCRs) sense changes in the extracellular pH to effect cell signaling for cellular homeostasis. They tend to be overexpressed in solid tumors associated with acidic extracellular pH, and are of direct interest as drug targets. How proton-sensing GPCRs sense extracellular acidification and activate upon protonation change is important to understand, because it may guide the design of therapeutics. Lack of publicly available experimental structures make it challenging to discriminate between conflicting mechanisms proposed for proton-binding, as main roles have been assigned to either an extracellular histidine cluster or to an internal carboxylic triad. Here we present a protocol to derive and evaluate structural models of the proton-sensing GPR68. This approach integrates state-of-the-art homology modeling with microsecond-timescale atomistic simulations, and with a detailed assessment of the compatibility of the structural models with known structural features of class A GPCRs. To decipher structural elements of potential interest for protonation-coupled conformational changes of GPR68, we used the best-compatible model as a starting point for independent atomistic simulations of GPR68 with different protonation states, and graph computations to characterize the response of GPR68 to changes in protonation. We found that GPR68 hosts an extended hydrogen-bond network that inter-connects the extracellular histidine cluster to the internal carboxylic triad, and which can even reach groups at the cytoplasmic G-protein binding site. Taken together, results suggest that GPR68 relies on dynamic, hydrogen-bond networks to inter-connect extracellular and internal proton-binding sites, and to elicit conformational changes at the cytoplasmic G-protein binding site.

Proton‐Sensing Receptor GPR65 Dynamically Traffics To and Signals From Multiple Cellular Compartments

Proton‐sensing G protein‐coupled receptors (GPCRs) allow cells to sense and respond to changes in physiological pH. Over the last 20 years, proton‐sensing membrane proteins have been implicated in a variety of biological functions and disease states, including inflammation, and stimulation and inhibition of oncogenic signaling pathways. Proton‐sensing GPCRs are unique in that, upon internalization and trafficking to acidic intracellular compartments, they are exposed to environments that are more likely to activate the receptor. However, little is known about whether and how proton‐sensing receptors signal from intracellular compartments. Characterizing proton‐sensing receptor trafficking and signaling is critical to understand how these receptors integrate responses from multiple locations in the cell, and how these responses change during the progression of diseases associated with acidic microenvironments. Here we use proton‐sensing receptor GPR65 as a prototype to study how receptor location influences proton‐sensing receptor signaling. We use live imaging of GPR65‐expressing cells to show that GPR65 internalizes from the plasma membrane at both neutral and acidic pH. Further, GPR65 localizes to early and late endosomes irrespective of extracellular pH. However, GPR65 increases second messenger cAMP by coupling to Gs proteins upon exposure to acidic extracellular pH. To determine whether GPR65 at internal compartments contributes to cAMP production, we pre‐treated GPR65‐expressing cells with Dyngo4a, an endocytosis inhibitor. GPR65 internalization was required for a full cAMP response after acidic extracellular pH. Together, our findings show that GPR65 dynamically traffics and signals from multiple cellular compartments irrespective of extracellular pH. Importantly, our results suggest that, unlike for most known GPCRs, activation of GPR65 is uncoupled from receptor trafficking.

GPR68 Inhibition with a Novel Group of Ogremorphin Inhibitors Upregulate Endothelial Barrier Function and Protect Against Bacterial Pathogens or Acidosis‐induced Inflammation in Lung Endothelium

BackgroundEndothelial dysfunction characterized by an increase in endothelial permeability and inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID‐19. Thus, drugs targeting the preservation and restoration of endothelial function represent an attractive therapeutic target to treat endothelial dysfunction‐derived cardiopulmonary diseases. G protein‐coupled receptors (GPCRs), especially a sub‐family of proton‐sensing GPCRs including GPR4 and GPR68, have been recently suggested to play a role in the modulation of endothelial function. In this study, we investigated the barrier protective and anti‐inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM‐1 in cultured human lung endothelial cells as well as in mice.MethodsEndothelial permeability was measured by monitoring transendothelial electrical resistance (TER) across human pulmonary arterial endothelial cells (HPAECs). Protein and mRNA expression levels of endothelial inflammation markers were analyzed by western blot and quantitative real time PCR, respectively. Acidosis was induced by switching cells to acidic pH (6.5) medium and luciferase‐based Tango assay was performed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heat‐killed Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples.ResultsBoth OGM8345 (1‐5 µM) and OGM‐1 (0.3‐1.5 µM) induced a robust dose‐dependent increase in basal EC barrier function that was evident by an 150‐200% increase in TER values. Both inhibitors also markedly attenuated LPS‐ and HKSA‐induced endothelial hyperpermeability. LPS or HKSA‐induced upregulation of inflammatory cytokines/chemokines genes TNF‐α, ICAM‐1, VCAM‐1, IL‐6, IL‐8, IL‐1β, and CXCL5 was significantly attenuated by OGMs as demonstrated by RT‐PCR analysis. Consistently, both OGMs suppressed LPS‐ and HKSA‐induced protein expression of phospho‐NFkB, VCAM‐1 and ICAM‐1. In contrast, pharmacologic inhibition of GPR4 by NE 52‐QQ57 failed to alleviate LPS or HKSA‐induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression as well as GPR68 activity that was inhibited by OGMs. Both OGMs attenuated vascular leak and lung inflammation caused by intratracheal injection of LPS or HKSA in C57BL/6 mice as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of inflammatory cells and protein content in bronchoalveolar lavage samples.ConclusionAltogether, these results establish an essential role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68‐selective inhibitors in improving endothelial dysfunction‐derived lung injuries.

Proton-Sensing GPCRs in Health and Disease.

The group of proton-sensing G-protein coupled receptors (GPCRs) consists of the four receptors GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132). These receptors are cellular sensors of acidification, a property that has been attributed to the presence of crucial histidine residues. However, the pH detection varies considerably among the group of proton-sensing GPCRs and ranges from pH of 5.5 to 7.8. While the proton-sensing GPCRs were initially considered to detect acidic cellular environments in the context of inflammation, recent observations have expanded our knowledge about their physiological and pathophysiological functions and many additional individual and unique features have been discovered that suggest a more differentiated role of these receptors in health and disease. It is known that all four receptors contribute to different aspects of tumor biology, cardiovascular physiology, and asthma. However, apart from their overlapping functions, they seem to have individual properties, and recent publications identify potential roles of individual GPCRs in mechanosensation, intestinal inflammation, oncoimmunological interactions, hematopoiesis, as well as inflammatory and neuropathic pain. Here, we put together the knowledge about the biological functions and structural features of the four proton-sensing GPCRs and discuss the biological role of each of the four receptors individually. We explore all currently known pharmacological modulators of the four receptors and highlight potential use. Finally, we point out knowledge gaps in the biological and pharmacological context of proton-sensing GPCRs that should be addressed by future studies.

Endocytic trafficking and signaling of proton‐sensing receptor GPR65

Proton-sensing G-protein coupled receptors (GPCRs) allow cells to sense and respond to changes in extracellular pH. Although pH disturbances have profound effects on cell function and disease progression, little is known about how proton-sensing GPCR activation, trafficking and signaling from distinct intracellular compartments contribute to cellular responses. Here we use GPR65, a proton-sensing GPCR that contributes to pathological conditions characterized by acidic tissue microenvironments such as cancer and chronic inflammatory conditions, as a prototype to study these questions. We use live imaging of HEK293 cells expressing tagged versions of our protein of interest, GPR65, to visualize receptor trafficking, sorting to intracellular compartments, and receptor activation using signaling and conformational biosensors in real time. Our preliminary results indicate that GPR65 localizes to intracellular compartments at steady state and recruits conformational biosensors to both the plasma membrane and intracellular compartments when exposed to neutral and acidic pH. Characterizing GPR65 trafficking and activation within the endosomal pathway will help us understand how proton-sensing receptors contribute to normal physiology and disease and provide new strategies to treat pathological conditions associated with acidic microenvironments.

The Molecular Evolution and Mechanism of Proton‐sensing GPCRs

Membrane receptors regulate many aspects of cell biology by enabling cells to sense and respond to their external environment. This is especially true for G protein-coupled receptors (GPCRs), capable of activating various intracellular signaling pathways upon stimulation by extracellular molecules ranging from small metabolites to large protein ligands. Three human GPCRs (GPR4, GPR65, and GPR68) are uniquely known for their proton (H+)-sensing capabilities, enabling the regulation of cell biology and physiology merely in response to acidosis. However, the mechanism underlying this proton sensing is poorly understood. In this study, we show these receptors evolved the ability to sense H+ by acquiring a triad of buried acidic residues. Using our informatics platform, pHinder, we initially identified this buried acidic triad as a distinct structural feature of proton-sensing GPCRs. Phylogenetics revealed the buried acidic triad emerged in GPR65, the ancestral proton-sensing receptor, and was maintained during the evolution of GPR4 and GPR68. To experimentally test the mechanistic importance of these triad residues, we developed Deep Variant Profiling (DVP), a method combining high-throughput CRISPR gene editing and GPCR signaling assays in yeast to profile large libraries of GPCR variants. Our results validated that most triad residues are the primary source of H+ sensing, and also show that Na+, an allosteric modulator of many other GPCRs, synergistically regulates H+ sensing by tuning the pKa values of triad residues within the physiological pH range. These findings reveal an evolutionarily supported mechanism of proton sensing by GPR4, GPR65, and GPR68, and also provide pH-insensitive variants for understanding how GPCR-based proton sensing regulates cell biology and physiology.

Roles of G protein-coupled receptors in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex disease with multiple pathogenic factors. Although the pathogenesis of IBD is still unclear, a current hypothesis suggests that genetic susceptibility, environmental factors, a dysfunctional immune system, the microbiome, and the interactions of these factors substantially contribute to the occurrence and development of IBD. Although existing and emerging drugs have been proven to be effective in treating IBD, none can cure IBD permanently. G protein-coupled receptors (GPCRs) are critical signaling molecules implicated in the immune response, cell proliferation, inflammation regulation and intestinal barrier maintenance. Breakthroughs in the understanding of the structures and functions of GPCRs have provided a driving force for exploring the roles of GPCRs in the pathogenesis of diseases, thereby leading to the development of GPCR-targeted medication. To date, a number of GPCRs have been shown to be associated with IBD, significantly advancing the drug discovery process for IBD. The associations between GPCRs and disease activity, disease severity, and disease phenotypes have also paved new avenues for the precise management of patients with IBD. In this review, we mainly focus on the roles of the most studied proton-sensing GPCRs, cannabinoid receptors, and estrogen-related GPCRs in the pathogenesis of IBD and their potential clinical values in IBD and some other diseases.

GPR31 and GPR151 are activated under acidic conditions.

Recent studies have revealed that not only proton-sensing channels, but also one family of G protein coupled-receptors (GPCRs) comprising OGR1, GPR4, G2A, and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31, and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31-Giα and GPR151-Giα fusion proteins in a narrow range between pH 6 and pH 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at approximately pH 5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML.Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function.In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway.In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. DisclosuresNo relevant conflicts of interest to declare.

Real-time monitoring of pH-dependent intracellular trafficking of ovarian cancer G protein-coupled receptor 1 in living leukocytes

G-protein coupled receptors (GPCRs) are involved in many diseases and important biological phenomena; elucidating the mechanisms underlying regulation of their signal transduction potentially provides both novel targets for drug discovery and insight into living systems. A proton-sensing GPCR, ovarian cancer G protein-coupled receptor 1 (OGR1), has been reported to be related to acidosis and diseases that cause tissue acidification, but the mechanism of proton-induced activation of OGR1-mediated signal transduction in acidic conditions remains unclear. Here, pH-dependent intracellular trafficking of OGR1 was visualized in living leukocytes by a real-time fluorescence microscopic method based on sortase A-mediated pulse labeling of OGR1. OGR1 labeled on the cell surface with a small fluorescent dye was clearly observed to remain in the plasma membrane during incubation in mildly acidic medium (pH 6.6) and to be internalized to the intracellular compartments on changing the medium to slightly basic pH (7.7). Quantitative single-cell image analysis showed that most of the internalized OGR1s were then recycled to the plasma membrane for signal transduction if the extracellular pH was returned to the mildly acidic state. However, in a minor population of cells (40%), the internalized OGR1s were retained in endosomes or transported to lysosomes and degraded, leading to low efficiency of their recycling to the plasma membrane. Thus, the present live-cell monitoring strongly suggests that the signal transduction activity of OGR1 is regulated by pH-dependent internalization and recycling to the plasma membrane.

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Compared with PSCs and PFs, CAFs have increased expression of GPR68 (a proton-sensing GPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Co-culture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.-Wiley, S. Z., Sriram, K., Liang, W., Chang, S. E., French, R., McCann, T., Sicklick, J., Nishihara, H., Lowy, A. M., Insel, P. A. GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

TDAG8 involved in initiating inflammatory hyperalgesia and establishing hyperalgesic priming in mice

Chronic pain, resulting from injury, arthritis, and cancer, is often accompanied by inflammation. High concentrations of protons found in inflamed tissues results in tissue acidosis, a major cause of pain and hyperalgesia. Acidosis signals may mediate a transition from acute to chronic hyperalgesia (hyperalgesic priming) via proton-sensing G-protein-coupled receptors (GPCRs). The expression of T-cell death-associated gene 8 (TDAG8), a proton-sensing GPCR, is increased during inflammatory hyperalgesia. Attenuating TDAG8 expression in the spinal cord inhibits bone cancer pain, but whether TDAG8 is involved in inflammatory hyperalgesia or hyperalgesic priming remains unclear. In this study, we used TDAG8-knockout or -knockdown to explore the role of TDAG8 in pain. Suppressed TDAG8 expression delayed the onset of inflammatory hyperalgesia and shortened hyperalgesic time in mice. In a dual acid-injection model (acid [pH 5.0] injected twice, 5 days apart), shRNA inhibition of TDAG8 shortened the duration of the second hyperalgesia. Similar results were found in TDAG8-deficient mice. The dual administration of TDAG8 agonist also confirmed that TDAG8 is involved in hyperalgsic priming. Accordingly, TDAG8 may mediate acidosis signals to initiate inflammatory hyperalgesia and establish hyperalgesic priming.

Proton-sensing G protein-coupled receptors as regulators of cell proliferation and migration during tumor growth and wound healing.

Dysregulation of pH is a feature of both tumor growth and tissue repair. In tumors, microenvironmental changes, like in lactate metabolism, lead to altered intra- and extracellular pH (pHi , pHe ) and vice versa. In wounds, barrier disruption results in extensive variations in pHe on the wound surface. It is known that altered extracellular proton concentrations have a major impact on cell turnover and migration as well as on the metabolic activity of cells involved in tumor spread and wound closure. The proton-sensing G protein-coupled receptors (GPCRs) GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A) are activated via a decrease in pHe and transduce this signal to molecular intracellular pathways. Based on the current knowledge, we speculate on the role of proton-sensing GPCRs in wound healing and on their potential as mechanistic linkers of tumor growth and tissue repair.

Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

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.

Role of extracellular proton-sensing OGR1 in regulation of insulin secretion and pancreatic β-cell functions

Insulin secretion with respect to pH environments has been investigated for a long time but its mechanism remains largely unknown. Extracellular pH is usually maintained at around 7.4 and, its change has been thought to occur in non-physiological situations. Acidification takes place under ischemic and inflammatory microenvironments, where stimulation of anaerobic glycolysis results in the production of lactic acid. In addition to ionotropic ion channels, such as transient receptor potential V1 (TRPV1) and acid-sensing ion channels (ASICs), metabotropic proton-sensing G protein-coupled receptors (GPCRs) have also been identified recently as proton-sensing machineries. While ionotropic ion channels usually sense strong acidic pH, proton-sensing GPCRs sense pH of 7.6 to 6.0 and have been shown to mediate a variety of biological actions in neutral and mildly acidic pH environments. Studies with receptor knockout mice have revealed that proton-sensing receptors, including ovarian cancer G protein-coupled receptor 1 (OGR1), a proton-sensing GPCRs, play a role in the regulation of insulin secretion and glucose metabolism under physiological conditions. Small molecule 3,5-disubstituted isoxazoles have recently been identified as OGR1 agonists working at neutral pH and have been shown to stimulate pancreatic β-cell differentiation and insulin synthesis. Thus, proton-sensing OGR1 may be an important player for insulin secretion and a potential target for improving β-cell function.

PH sensing and regulation in cancer

Cells maintain intracellular pH (pHi) within a narrow range (7.1–7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H+-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.

Acidic tumor microenvironment and pH-sensing G protein-coupled receptors

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

Expression and Function of Proton-Sensing G-Protein-Coupled Receptors in Inflammatory Pain

BackgroundChronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Despite the availability of pharmacologic treatments, chronic inflammatory pain remains inadequately treated. Understanding the nociceptive signaling pathways of such pain is therefore important in developing long-acting treatments with limited side effects. High local proton concentrations (tissue acidosis) causing direct excitation or modulation of nociceptive sensory neurons by proton-sensing receptors are responsible for pain in some inflammatory pain conditions. We previously found that all four proton-sensing G-protein-coupled receptors (GPCRs) are expressed in pain-relevant loci (dorsal root ganglia, DRG), which suggests their possible involvement in nociception, but their functions in pain remain unclear.ResultsIn this study, we first demonstrated differential change in expression of proton-sensing GPCRs in peripheral inflammation induced by the inflammatory agents capsaicin, carrageenan, and complete Freund's adjuvant (CFA). In particular, the expression of TDAG8, one proton-sensing GPCR, was increased 24 hours after CFA injection because of increased number of DRG neurons expressing TDAG8. The number of DRG neurons expressing both TDAG8 and transient receptor potential vanilloid 1 (TRPV1) was increased as well. Further studies revealed that TDAG8 activation sensitized the TRPV1 response to capsaicin, suggesting that TDAG8 could be involved in CFA-induced chronic inflammatory pain through regulation of TRPV1 function.ConclusionEach subtype of the OGR1 family was expressed differently, which may reflect differences between models in duration and magnitude of hyperalgesia. Given that TDAG8 and TRPV1 expression increased after CFA-induced inflammation and that TDAG8 activation can lead to TRPV1 sensitization, it suggests that high concentrations of protons after inflammation may not only directly activate proton-sensing ion channels (such as TRPV1) to cause pain but also act on proton-sensing GPCRs to regulate the development of hyperalgesia.

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E(2)-induced production of cAMP were significantly decreased. A remarkable increase in [(35)S]guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G(i/o) proteins. Involvement of GPR20 in the activation of G(i/o) proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [(35)S]GTPgammaS incorporation and inhibition of the prostaglandin E(2)-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [(3)H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G(i) proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling.