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Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell-cycle progression and proliferation compared with WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared with WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathological phenotypic switching and proliferation in VSM. NEW & NOTEWORTHY: Extracellular acidosis remains an understudied feature of many pathologies. We examined a potential regulatory role for pH-sensing GPR68 in vascular smooth muscle (VSM) growth in the context of CVD. With in vivo and in vitro growth models with GPR68-deficient mice and GPR68 induction strategies, novel findings revealed capacity of GPR68 to attenuate growth through the small GTPase Rap1A. These observations highlight GPR68 and its effector Rap1A as possible therapeutic targets to combat pathological VSM growth. [ABSTRACT FROM AUTHOR]

The precise regulation of pH homeostasis is crucial for normal physiology. However, in tissue microenvironments, it can be impacted by pathological conditions such as inflammation and cancer. Due to the overproduction and accumulation of acids (protons), the extracellular pH is characteristically more acidic in inflamed tissues and tumors in comparison to normal tissues. A family of proton-sensing G-protein-coupled receptors (GPCRs) has been identified as molecular sensors for cells responding to acidic tissue microenvironments. Herein, we review the current research progress pertaining to these proton-sensing GPCRs, including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), in inflammation and cancer. Growing evidence suggests that GPR4 and GPR68 are mainly pro-inflammatory, whereas GPR65 is primarily anti-inflammatory, in various inflammatory disorders. Both anti- and pro-tumorigenic effects have been reported for this family of receptors. Moreover, antagonists and agonists targeting proton-sensing GPCRs have been developed and evaluated in preclinical models. Further research is warranted to better understand the roles of these proton-sensing GPCRs in pathophysiology and is required in order to exploit them as potential therapeutic targets for disease treatment. [ABSTRACT FROM AUTHOR]

Abstract Objective Inhibition and knockdown of GPR68 negatively affects glioblastoma cell survival in vitro by inducing ferroptosis. Herein, we aimed to demonstrate that inhibition of GPR68 reduces the survival of glioblastoma cells in vivo using two orthotopic larval xenograft models in Danio rerio, using GBM cell lines U87-MG and U138-MG. In vivo survival of the cancer cells was assessed in the setting of GPR68 inhibition or knockdown. Results In vitro, shRNA-mediated knockdown of GPR68 inhibition demonstrated potent cytotoxic effects against U87 and U138 glioblastoma cell lines. This effect was associated with increased intracellular lipid peroxidation, suggesting ferroptosis as the underlying mechanism of cell death. Translating these findings in vivo, we established a novel xenograft model in zebrafish by successfully grafting fluorescently labeled human glioblastoma cells, which were previously shown to overexpress GPR68. shRNA knockdown of GPR68 significantly reduced the viability of grafted GBM cells within this model. Additionally, treatment with ogremorphin (OGM), a highly specific small molecule inhibitor of GPR68, also reduced the viability of grafted GBM cells with limited toxicity to the developing zebrafish embryos. This study suggests that therapeutic targeting of GPR68 with small molecules like OGM represents a promising approach for the treatment of GBM.

Objective: Inhibition and knockdown of GPR68 negatively affects glioblastoma cell survival in vitro by inducing ferroptosis. Herein, we aimed to demonstrate that inhibition of GPR68 reduces the survival of glioblastoma cells in vivo using two orthotopic larval xenograft models in Danio rerio, using GBM cell lines U87-MG and U138-MG. In vivo survival of the cancer cells was assessed in the setting of GPR68 inhibition or knockdown. Results: In vitro, shRNA-mediated knockdown of GPR68 inhibition demonstrated potent cytotoxic effects against U87 and U138 glioblastoma cell lines. This effect was associated with increased intracellular lipid peroxidation, suggesting ferroptosis as the underlying mechanism of cell death. Translating these findings in vivo, we established a novel xenograft model in zebrafish by successfully grafting fluorescently labeled human glioblastoma cells, which were previously shown to overexpress GPR68. shRNA knockdown of GPR68 significantly reduced the viability of grafted GBM cells within this model. Additionally, treatment with ogremorphin (OGM), a highly specific small molecule inhibitor of GPR68, also reduced the viability of grafted GBM cells with limited toxicity to the developing zebrafish embryos. This study suggests that therapeutic targeting of GPR68 with small molecules like OGM represents a promising approach for the treatment of GBM. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia , inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment's osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. Methods: We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. Results: The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Conclusions: Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA. [ABSTRACT FROM AUTHOR]

Abstract Background Dose-limiting toxicities significantly impact the benefit/risk profile of many drugs. Whole genome sequencing (WGS) in patients receiving drugs with dose-limiting toxicities can identify therapeutic hypotheses to prevent these toxicities. Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting neurological toxicity of chemotherapies with no effective approach for prevention. Methods We conducted a genetic study of time-to-first peripheral neuropathy event using 30× germline WGS data from whole blood samples from 4900 European-ancestry cancer patients in 14 randomized controlled trials. A substantial number of patients in these trials received taxane and platinum-based chemotherapies as part of their treatment regimen, either standard of care or in combination with the PD-L1 inhibitor atezolizumab. The trials spanned several cancers including renal cell carcinoma, triple negative breast cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, and melanoma. Results We identified a locus consisting of low-frequency variants in intron 13 of GRID2 associated with time-to-onset of first peripheral neuropathy (PN) indexed by rs17020773 (p = 2.03 × 10−8, all patients, p = 6.36 × 10−9, taxane treated). Gene-level burden analysis identified rare coding variants associated with increased PN risk in the C-terminus of GPR68 (p = 1.59 × 10−6, all patients, p = 3.47 × 10−8, taxane treated), a pH-sensitive G-protein coupled receptor (GPCR). The variants driving this signal were found to alter predicted arrestin binding motifs in the C-terminus of GPR68. Analysis of snRNA-seq from human dorsal root ganglia (DRG) indicated that expression of GPR68 was highest in mechano-thermo-sensitive nociceptors. Conclusions Our genetic study provides insight into the impact of low-frequency and rare coding genetic variation on PN risk and suggests that further study of GPR68 in sensory neurons may yield a therapeutic hypothesis for prevention of CIPN.

Background Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment’s osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. Methods We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. Results The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Conclusions Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA.

Background: Dose-limiting toxicities significantly impact the benefit/risk profile of many drugs. Whole genome sequencing (WGS) in patients receiving drugs with dose-limiting toxicities can identify therapeutic hypotheses to prevent these toxicities. Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting neurological toxicity of chemotherapies with no effective approach for prevention. Methods: We conducted a genetic study of time-to-first peripheral neuropathy event using 30× germline WGS data from whole blood samples from 4900 European-ancestry cancer patients in 14 randomized controlled trials. A substantial number of patients in these trials received taxane and platinum-based chemotherapies as part of their treatment regimen, either standard of care or in combination with the PD-L1 inhibitor atezolizumab. The trials spanned several cancers including renal cell carcinoma, triple negative breast cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, and melanoma. Results: We identified a locus consisting of low-frequency variants in intron 13 of GRID2 associated with time-to-onset of first peripheral neuropathy (PN) indexed by rs17020773 (p = 2.03 × 10−8, all patients, p = 6.36 × 10−9, taxane treated). Gene-level burden analysis identified rare coding variants associated with increased PN risk in the C-terminus of GPR68 (p = 1.59 × 10−6, all patients, p = 3.47 × 10−8, taxane treated), a pH-sensitive G-protein coupled receptor (GPCR). The variants driving this signal were found to alter predicted arrestin binding motifs in the C-terminus of GPR68. Analysis of snRNA-seq from human dorsal root ganglia (DRG) indicated that expression of GPR68 was highest in mechano-thermo-sensitive nociceptors. Conclusions: Our genetic study provides insight into the impact of low-frequency and rare coding genetic variation on PN risk and suggests that further study of GPR68 in sensory neurons may yield a therapeutic hypothesis for prevention of CIPN. [ABSTRACT FROM AUTHOR]

Introduction: Melanoma is a common and aggressive type of skin cancer with rising incidence rate globally. Gender is one of the determining factors, and overall males have a higher risk of developing melanoma as well as worse prognosis. Emerging evidence show that GPR68, a G protein-coupled receptor that is sensitive to acid and mechanical stimulations for cellular microenvironment, plays an important role in tumor biology. However, whether GPR68 is involved in gender-dependent regulation of tumor growth is unclear. Methods: We established a syngeneic melanoma model in Gpr68-deficient mice and investigated tumor growth in males and females. The GPR68 activation-induced cellular responses of melanocytes, including intracellular calcium dynamics, proliferation and migration were measured. The landscape of tumor-infiltrating immune cells were analyzed by flow cytometry and the expression various cytokines were checked by qRT-PCR. Results: GPR68 is required for melanoma growth in males but dispensable in females. GPR68 is expressed and functional in B16-F10 melanocytes, but the activity of the receptor does not directly contribute to proliferation and migration of the cells. GPR68 inhibits infiltration of CD45+ lymphocytes, CD8+ T cells and NK cells in melanoma in male mice, but has no apparent effect in females. Furthermore, GPR68 functionally inhibits the expression of IFNγ in the tumor infiltrating CD8+ T cells and NK cells as well as the inflammatory cytokine expression in the spleen in male mice but not in females. Our results show the gender-dependent modulatory effect of GPR68 on tumor-infiltrating immune cells and their tumor-killing capacity. Discussion: GPR68 is sensor for acid and mechanical stimulations, which are two important factors in the microenvironment associated with tumor growth and metastasis. Our results suggest a prominent role of the receptor molecules in tumor biology in a gender-dependent manner. Since GPCRs are more feasible to develop small molecule drugs compared to transcription factors, our study demonstrates the potential of GPR68 as a novel druggable therapeutic target for melanoma in male patients. [ABSTRACT FROM AUTHOR]

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.

Abstract Background We have previously reported that a new intronless gene for casein kinase 2α (CK2α), CSNK2A3, is expressed in human cells. The promoter of the well-known CK2α, CSNK2A1, displays characteristics of a housekeeping gene, whereas CSNK2A3 has a characteristic of a regulated promoter with two TATA boxes and a CAAT box. GPR68, a family of the G protein-coupled receptors, is also known as ovarian cancer G protein-coupled receptor 1 (OGR1). In the current study, we analyzed the roles of CK2α genes and neutral endopeptidase (NEP), a key enzyme that influences a variety of malignancies, in the OGR1-induced inhibition of A549 cell migration. Methods We analyzed the transcript expressions of both the CK2α genes (CSNK2A1 and CSNK2A3) and NEP upon OGR1 overexpression. Protein expression of CK2α and NEP were also analyzed. We further elucidated the functional roles of both CK2α and NEP in the OGR1-induced inhibition of A549 cell migration in vitro using a wound-healing assay. We also analyzed the molecular mechanisms involved in the OGR1-induced inhibition of lung cancer cell migration. Results The findings of this study showed that OGR1 upregulated the expression of CSNK2A3 but not CSNK2A1 in the A549 cells. The findings further suggested OGR1 also upregulates the expression of NEP. The OGR1-induced inhibition of A549 cell migration was abrogated completely by inhibition of CK2α activity, whereas partial abrogation (~ 30%) was observed in the presence of NEP inhibition. The results also revealed that OGR1 regulates CSNK2A3 via activation of Rac1/cdc42 and MAPKs pathways. CK2 is ubiquitously expressed, and in contrast, is believed to be a constitutively active enzyme, and its regulation appears to be independent of known second messengers. Conclusion In the current study, we report for the first time the OGR1-induced regulation of CSNK2A3, CK2αP, and NEP in A549 cancer cells. Our study also decoded the downstream cellular proteins of OGR1 as well as the molecular mechanism involved in OGR1-induced inhibition of A549 cell migration. The findings of this research suggest the potential therapeutic targets to inhibit lung cancer progression.

Von Willebrand Factor (VWF) can promote platelet adhesion to the post-atherosclerotic regions to initiate thrombosis. The synthesis and secretion of VWF are important functions of endothelial cells (ECs). However, the mechanism through which blood flow regulates endothelial secretion of VWF remains unclear. We utilized a parallel-plate flow apparatus to apply fluid shear stress to human umbilical vein endothelial cells (HUVECs). Compared with pulsatile shear stress that mimics laminar flow in the straight parts of arteries or upstream of atherosclerotic stenosis sites, short-term exposure to oscillatory shear stress (OS) that mimics disturbed flow increased VWF secretion independent of affecting synaptosomal-associated protein 23 (SNAP23) expression and promoted the translocation of SNAP23 to the cell membrane. Vimentin associated with SNAP23, and this association was enhanced by OS or histamine. Acrylamide, a reagent that disrupts vimentin intermediate filaments, prevented histamine/OS-induced SNAP23 translocation, as well as VWF secretion. Immunofluorescence analysis revealed that the polarity of the vimentin intermediate filament network decreased after stimulation with histamine or OS. In addition, inhibition of protein kinase A (PKA) or G protein coupled receptor 68 (GPR68) eliminated the histamine/OS-induced phosphorylation of vimentin at Ser38 and secretion of VWF. Furthermore, syntaxin 7 might assist with the translocation of SNAP23 to the cell membrane, thus playing a role in promoting VWF secretion. The GPR68/PKA/vimentin signaling pathway may represent a novel mechanism for the regulation of SNAP23-mediated VWF secretion by ECs under OS and provide strategies for the prevention of atherosclerosis-related thrombosis. • Explore new mechanism through which blood flow regulates endothelial VWF secretion. • Short OS promotes VWF secretion through vimentin-associated translocation of SNAP23. • Syntaxin 7 interacts with vimentin to mediate the transport of SNAP23. • The GPR68/PKA/vimentin pathway involved in shear stress-induced VWF secretion. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly invasive cancers have disrupted metabolism that leads to the creation of an acidic tumor microenvironment. Among the proton-sensing G protein-coupled receptors is GPR68. In this study, we aimed to explore the expression pattern of GPR68 in tissues from BC patients as well as different BC cell lines. Methods: In-silico tools were used to assess the expression of GPR68 in BC patients. The expression pattern was validated in fresh and paraffin-embedded sections of BC patients using qPCR and immunohistochemistry (IHC), respectively. Also, in-silico tools investigated GPR68 expression in different BC cell lines. Validation of GPR68 expression was performed using qPCR and immunofluorescence techniques in four different BC cell lines (MCF-7, MDA-MB-231, BT-549 and SkBr3). Results: GPR68 expression was found to be significantly increased in BC patients using the in-silico tools and validation using qPCR and IHC. Upon classification according to the molecular subtypes, the luminal subtype showed the highest GPR68 expression followed by triple-negative and Her2-enriched cells. However, upon validation in the recruited cohort, the triple-negative molecular subtype of BC patients showed the highest GPR68 expression. Also, in-silico and validation data revealed that the triple-negative breast cancer cell line MDA-MB-231 showed the highest expression of GPR68. Conclusion: Therefore, this study highlights the potential utilization of GPR68 as a possible diagnostic and/or prognostic marker in BC. [ABSTRACT FROM AUTHOR]

BackgroundBreast cancer (BC) is the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly invasive cancers have disrupted metabolism that leads to the creation of an acidic tumor microenvironment. Among the proton-sensing G protein-coupled receptors is GPR68. In this study, we aimed to explore the expression pattern of GPR68 in tissues from BC patients as well as different BC cell lines. Methods: In-silico tools were used to assess the expression of GPR68 in BC patients. The expression pattern was validated in fresh and paraffin-embedded sections of BC patients using qPCR and immunohistochemistry (IHC), respectively. Also, in-silico tools investigated GPR68 expression in different BC cell lines. Validation of GPR68 expression was performed using qPCR and immunofluorescence techniques in four different BC cell lines (MCF-7, MDA-MB-231, BT-549 and SkBr3). Results: GPR68 expression was found to be significantly increased in BC patients using the in-silico tools and validation using qPCR and IHC. Upon classification according to the molecular subtypes, the luminal subtype showed the highest GPR68 expression followed by triple-negative and Her2-enriched cells. However, upon validation in the recruited cohort, the triple-negative molecular subtype of BC patients showed the highest GPR68 expression. Also, in-silico and validation data revealed that the triple-negative breast cancer cell line MDA-MB-231 showed the highest expression of GPR68. Conclusion: Therefore, this study highlights the potential utilization of GPR68 as a possible diagnostic and/or prognostic marker in BC.

Background: We have previously reported that a new intronless gene for casein kinase 2α (CK2α), CSNK2A3, is expressed in human cells. The promoter of the well-known CK2α, CSNK2A1, displays characteristics of a housekeeping gene, whereas CSNK2A3 has a characteristic of a regulated promoter with two TATA boxes and a CAAT box. GPR68, a family of the G protein-coupled receptors, is also known as ovarian cancer G protein-coupled receptor 1 (OGR1). In the current study, we analyzed the roles of CK2α genes and neutral endopeptidase (NEP), a key enzyme that influences a variety of malignancies, in the OGR1-induced inhibition of A549 cell migration.Methods: We analyzed the transcript expressions of both the CK2α genes (CSNK2A1 and CSNK2A3) and NEP upon OGR1 overexpression. Protein expression of CK2α and NEP were also analyzed. We further elucidated the functional roles of both CK2α and NEP in the OGR1-induced inhibition of A549 cell migration in vitro using a wound-healing assay. We also analyzed the molecular mechanisms involved in the OGR1-induced inhibition of lung cancer cell migration.Results: The findings of this study showed that OGR1 upregulated the expression of CSNK2A3 but not CSNK2A1 in the A549 cells. The findings further suggested OGR1 also upregulates the expression of NEP. The OGR1-induced inhibition of A549 cell migration was abrogated completely by inhibition of CK2α activity, whereas partial abrogation (~ 30%) was observed in the presence of NEP inhibition. The results also revealed that OGR1 regulates CSNK2A3 via activation of Rac1/cdc42 and MAPKs pathways. CK2 is ubiquitously expressed, and in contrast, is believed to be a constitutively active enzyme, and its regulation appears to be independent of known second messengers.Conclusion: In the current study, we report for the first time the OGR1-induced regulation of CSNK2A3, CK2αP, and NEP in A549 cancer cells. Our study also decoded the downstream cellular proteins of OGR1 as well as the molecular mechanism involved in OGR1-induced inhibition of A549 cell migration. The findings of this research suggest the potential therapeutic targets to inhibit lung cancer progression. [ABSTRACT FROM AUTHOR]

The proton-sensing receptor, ovarian cancer G protein-coupled receptor (OGR1), has been shown to be expressed in airway smooth muscle (ASM) cells and is capable of promoting ASM contraction in response to decreased extracellular pH. OGR1 knockout (OGR1KO) mice are reported to be resistant to the asthma features induced by inhaled allergen. We recently described certain benzodiazepines as OGR1 activators capable of mediating both procontractile and prorelaxant signaling in ASM cells. Here we assess the effect of treatment with the benzodiazepines lorazepam or sulazepam on the asthma phenotype in wildtype (WT) and OGR1KO mice subjected to inhaled house dust mite (HDM; Dermatophagoides pteronyssius) challenge for 3 wk. In contrast to previously published reports, both WT and OGR1KO mice developed significant allergen-induced lung inflammation and airway hyperresponsiveness (AHR). In WT mice, treatment with sulazepam (a Gs-biased OGR1 agonist), but not lorazepam (a balanced OGR1 agonist), prevented allergen-induced AHR, although neither drug inhibited lung inflammation. The protection from development of AHR conferred by sulazepam was absent in OGR1KO mice. Treatment of WT mice with sulazepam also resulted in significant inhibition of HDM-induced collagen accumulation in the lung tissue. These findings suggest that OGR1 expression is not a requirement for development of the allergen-induced asthma phenotype, but OGR1 can be targeted by the Gs-biased OGR1 agonist sulazepam (but not the balanced agonist lorazepam) to protect from allergen-induced AHR, possibly mediated via suppression of chronic bronchoconstriction and airway remodeling in the absence of effects on airway inflammation. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All the authors have read and approved the submission for publication., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract α1A‐ and α1B‐adrenoceptors (ARs) are G protein‐coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A‐ and α1B‐AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1‐ARs. Here, using thermostabilized, purified, α1A‐ and α1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A‐ and α1B‐ARs. Similarly, in cell‐based assays expressing unmodified α1A‐ and α1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A‐ and α1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

• Mechanical stress and extracellular acidosis are features of solid cancers. • They also promote cancer development and metastatic spread. • Dysregulated intracellular Ca2+ signals are equally typical for and advance cancer. • Some Ca2+ mobilising ion channels and receptors are both mechano- and pH-sensitive. • These proteins are promising therapeutic targets for combating cancer. Alterations in the (bio)chemical and physical microenvironment of cells accompany and often promote disease formation and progression. This is particularly well established for solid cancers, which are typically stiffer than the healthy tissue in which they arise, and often display profound acidification of their interstitial fluid. Cell surface receptors can sense changes in the mechanical and (bio)chemical properties of the surrounding extracellular matrix and fluid, and signalling through these receptors is thought to play a key role in disease development and advancement. This review will look at ion channels and G protein coupled receptors that are activated by mechanical cues and extracellular acidosis, and stimulation of which results in increases in intracellular Ca2+ concentrations. Cellular Ca2+ levels are dysregulated in cancer as well as cancer-associated cells, and mechano- and proton-sensing proteins likely contribute to these aberrant intracellular Ca2+ signals, making them attractive targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

α1A‐ and α1B‐adrenoceptors (ARs) are G protein‐coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A‐ and α1B‐AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1‐ARs. Here, using thermostabilized, purified, α1A‐ and α1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A‐ and α1B‐ARs. Similarly, in cell‐based assays expressing unmodified α1A‐ and α1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A‐ and α1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines. [ABSTRACT FROM AUTHOR]

Benzodiazepines (BZDs) are commonly prescribed for pancreatic cancer patients. To investigate the correlation between BZDs and survival outcomes a recent study by Cornwell et al. found that lorazepam (LOR) correlates with poor survival. The mechanistic study shows that LOR increases interleukin 6 (IL6) expression in cancer-associated fibroblasts via GPR68., Competing Interests: Declaration of interests None are declared by the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 (GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the ?-aminobutyric acid A (GABAA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial. [ABSTRACT FROM AUTHOR]

Introduction: Melanoma is a common and aggressive type of skin cancer with rising incidence rate globally. Gender is one of the determining factors, and overall males have a higher risk of developing melanoma as well as worse prognosis. Emerging evidence show that GPR68, a G protein-coupled receptor that is sensitive to acid and mechanical stimulations for cellular microenvironment, plays an important role in tumor biology. However, whether GPR68 is involved in gender-dependent regulation of tumor growth is unclear., Methods: We established a syngeneic melanoma model in Gpr68 -deficient mice and investigated tumor growth in males and females. The GPR68 activation-induced cellular responses of melanocytes, including intracellular calcium dynamics, proliferation and migration were measured. The landscape of tumor-infiltrating immune cells were analyzed by flow cytometry and the expression various cytokines were checked by qRT-PCR., Results: GPR68 is required for melanoma growth in males but dispensable in females. GPR68 is expressed and functional in B16-F10 melanocytes, but the activity of the receptor does not directly contribute to proliferation and migration of the cells. GPR68 inhibits infiltration of CD45 + lymphocytes, CD8 + T cells and NK cells in melanoma in male mice, but has no apparent effect in females. Furthermore, GPR68 functionally inhibits the expression of IFNγ in the tumor infiltrating CD8 + T cells and NK cells as well as the inflammatory cytokine expression in the spleen in male mice but not in females. Our results show the gender-dependent modulatory effect of GPR68 on tumor-infiltrating immune cells and their tumor-killing capacity., Discussion: GPR68 is sensor for acid and mechanical stimulations, which are two important factors in the microenvironment associated with tumor growth and metastasis. Our results suggest a prominent role of the receptor molecules in tumor biology in a gender-dependent manner. Since GPCRs are more feasible to develop small molecule drugs compared to transcription factors, our study demonstrates the potential of GPR68 as a novel druggable therapeutic target for melanoma in male patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ye, Zhu, Zhong, Song, Li, Xiao, Huang, Zhang, Wu, Zhang, Xiang and Xu.)

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation. [ABSTRACT FROM AUTHOR]

Diabetes (either type 1 or type 2) is due to insufficient functional β-cell mass. Research has, therefore, aimed to discover new ways to maintain or increase either β-cell mass or function. For this purpose, rodents have mainly been used as model systems and a large number of discoveries have been made. Meanwhile, although we have learned that rodent models represent powerful systems to model β-cell development, function and destruction, we realize that there are limitations when attempting to transfer the data to what is occurring in humans. Indeed, while human β-cells share many similarities with rodent β-cells, they also differ on a number of important parameters. In this context, developing ways to study human β-cell development, function and death represents an important challenge. This review will describe recent data on the development and use of convenient sources of human β-cells that should be useful tools to discover new ways to modulate functional β-cell mass in humans. [ABSTRACT FROM AUTHOR]

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin ( Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. EXPERIMENTAL APPROACH Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. KEY RESULTS ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar 'dose'-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. CONCLUSIONS AND IMPLICATIONS These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases. [ABSTRACT FROM AUTHOR]

Ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) is a member of proton-sensing G-protein-coupled receptors, involved in cardiovascular physiology, tumor biology, and asthma, and exerts a neuroprotective effect against brain ischemia. The effects of GPR68 on anesthesia-induced nerve damage and myelination were investigated in this study. First, 2-day old postnatal rats were exposed to 4.9% sevoflurane for 2 h. Data from hematoxylin and eosin staining and Nissl staining showed that sevoflurane induced pathological changes in the hippocampus with a reduced number of neurons. GPR68 was downregulated in the hippocampus of sevoflurane-induced rats. Second, sevoflurane-induced rats were injected with adeno-associated virus (AAV)-mediated overexpression of GPR68, and overexpression of GPR68 ameliorated sevoflurane-induced pathological changes, enhanced the number of neurons, and improved the learning and memory function. Moreover, overexpression of GPR68 increased the number of BrdU-positive and Olig2-positive cells and enhanced protein expression of Olig2 in sevoflurane-induced rats. Third, the number of myelin basic protein (MBP) positive cells and protein expression of MBP in sevoflurane-induced rats were also enhanced by injection with AAV-GPR68. Overexpression of GPR68 attenuated sevoflurane-induced neuronal apoptosis and oxidative stress in rats. Lastly, overexpression of GPR68 upregulated protein expression of the brain-derived neurotrophic factor (BDNF) by increasing cAMP and phosphorylated cAMP response element-binding protein (CREB). In conclusion, GPR68 alleviated sevoflurane-induced nerve damage and myelination through BDNF-mediated activation of the cAMP/CREB pathway.

Brain acid signaling plays important roles in both physiological and disease conditions. One key neuronal metabotropic proton receptor in the brain is GPR68, which contributes to hippocampal long-term potentiation (LTP) and mediates neuroprotection in acidotic and ischemic conditions. Here, to gain greater understanding of GPR68 function in the brain, we performed mRNA-Seq analysis in mice. First, we studied sham-operated animals to determine baseline expression. Compared to wild type (WT), GPR68-/- (KO) brain downregulated genes that are enriched in Gene Ontology (GO) terms of misfolding protein binding, response to organic cyclic compounds, and endoplasmic reticulum chaperone complex. Next, we examined the expression profile following transient middle cerebral artery occlusion (tMCAO). tMCAO-upregulated genes cluster to cytokine/chemokine-related functions and immune responses, while tMCAO-downregulated genes cluster to channel activities and synaptic signaling. For proton-sensitive receptors, tMCAO downregulated ASIC1a and upregulated GPR4 and GPR65, but had no effect on ASIC2, PAC, or GPR68. GPR68 deletion did not alter the expression of these proton receptors, either at baseline or after ischemia. Lastly, we performed GeneVenn analysis of differential genes at baseline and post-tMCAO. Ischemia upregulated the expression of three hemoglobin genes, along with H2-Aa, Ppbp, Siglece, and Tagln, in WT but not in KO. Immunostaining showed that tMCAO-induced hemoglobin localized to neurons. Western blot analysis further showed that hemoglobin induction is GPR68-dependent. Together, these data suggest that GPR68 deletion at baseline disrupts chaperone functions and cellular signaling responses and imply a contribution of hemoglobin-mediated antioxidant mechanism to GPR68-dependent neuroprotection in ischemia. [ABSTRACT FROM AUTHOR]

• The ability to sense mechanical stimuli is a well-established feature of a number of G protein coupled receptors (GPCRs). • The underlying structural components are unclear. • A recent paper provides compelling evidence for essential involvement of the GPCR helix 8 motif in this process. • This paper represents a significant and critical increase in our understanding how mechanical stimuli are sensed by GPCRs. [ABSTRACT FROM AUTHOR]

Brain acid signaling plays important roles in both physiological and disease conditions. One key neuronal metabotropic proton receptor in the brain is GPR68, which contributes to hippocampal long-term potentiation (LTP) and mediates neuroprotection in acidotic and ischemic conditions. Here, to gain greater understanding of GPR68 function in the brain, we performed mRNA-Seq analysis in mice. First, we studied sham-operated animals to determine baseline expression. Compared to wild type (WT), GPR68-/- (KO) brain downregulated genes that are enriched in Gene Ontology (GO) terms of misfolding protein binding, response to organic cyclic compounds, and endoplasmic reticulum chaperone complex. Next, we examined the expression profile following transient middle cerebral artery occlusion (tMCAO). tMCAO-upregulated genes cluster to cytokine/chemokine-related functions and immune responses, while tMCAO-downregulated genes cluster to channel activities and synaptic signaling. For proton-sensitive receptors, tMCAO downregulated ASIC1a and upregulated GPR4 and GPR65, but had no effect on ASIC2, PAC, or GPR68. GPR68 deletion did not alter the expression of these proton receptors, either at baseline or after ischemia. Lastly, we performed GeneVenn analysis of differential genes at baseline and post-tMCAO. Ischemia upregulated the expression of three hemoglobin genes, along with H2-Aa, Ppbp, Siglece, and Tagln, in WT but not in KO. Immunostaining showed that tMCAO-induced hemoglobin localized to neurons. Western blot analysis further showed that hemoglobin induction is GPR68-dependent. Together, these data suggest that GPR68 deletion at baseline disrupts chaperone functions and cellular signaling responses and imply a contribution of hemoglobin-mediated antioxidant mechanism to GPR68-dependent neuroprotection in ischemia., (© 2021 Federation of American Societies for Experimental Biology.)

Summary The physical environment critically affects cell shape, proliferation, differentiation, and survival by exerting mechanical forces on cells. These forces are sensed and transduced into intracellular signals and responses by cells. A number of different membrane and cytoplasmic proteins have been implicated in sensing mechanical forces, but the picture is far from complete, and the exact transduction pathways remain largely elusive. Furthermore, mechanosensation takes place alongside chemosensation, and cells need to integrate physical and chemical signals to respond appropriately and ensure normal tissue and organ development and function. Here, we report that ovarian cancer G protein coupled receptor 1 (OGR1) (aka GPR68) acts as coincidence detector of membrane stretch and its physiological ligand, extracellular H+. Using fluorescence imaging, substrates of different stiffness, microcontact printing methods, and cell-stretching techniques, we show that OGR1 only responds to extracellular acidification under conditions of membrane stretch and vice versa. The level of OGR1 activity mirrors the extent of membrane stretch and degree of extracellular acidification. Furthermore, actin polymerization in response to membrane stretch is critical for OGR1 activity, and its depolymerization limits how long OGR1 remains responsive following a stretch event, thus providing a "memory" for past stretch. Cells experience changes in membrane stretch and extracellular pH throughout their lifetime. Because OGR1 is a widely expressed receptor, it represents a unique yet widespread mechanism that enables cells to respond dynamically to mechanical and pH changes in their microenvironment by integrating these chemical and physical stimuli at the receptor level. Highlights • OGR1 (aka GPR68) requires cell stretch and extracellular protons to activate • Cell stretch and extracellular acidosis synergistically promote OGR1 signaling • Cell-stretch-mediated actin polymerization is crucial for OGR1 activity • Actin depolymerization limits how long OGR1 stays responsive after cell stretch Wei et al. show that the proton-sensing receptor OGR1/GPR68 is a coincidence detector of extracellular pH and substrate stiffness via changes in cell stretch. Changes in matrix stiffness and pH occur physiologically and pathologically. OGR1 is ideally positioned to sense and respond to changes in both, thus influencing cell, tissue, and organ fate. [ABSTRACT FROM AUTHOR]

Alterations in the (bio)chemical and physical microenvironment of cells accompany and often promote disease formation and progression. This is particularly well established for solid cancers, which are typically stiffer than the healthy tissue in which they arise, and often display profound acidification of their interstitial fluid. Cell surface receptors can sense changes in the mechanical and (bio)chemical properties of the surrounding extracellular matrix and fluid, and signalling through these receptors is thought to play a key role in disease development and advancement. This review will look at ion channels and G protein coupled receptors that are activated by mechanical cues and extracellular acidosis, and stimulation of which results in increases in intracellular Ca 2+ concentrations. Cellular Ca 2+ levels are dysregulated in cancer as well as cancer-associated cells, and mechano- and proton-sensing proteins likely contribute to these aberrant intracellular Ca 2+ signals, making them attractive targets for therapeutic intervention., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

α 1A - and α 1B -adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α 1A - and α 1B -AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α 1 -ARs. Here, using thermostabilized, purified, α 1A - and α 1B -ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α 1A - and α 1B -ARs. Similarly, in cell-based assays expressing unmodified α 1A - and α 1B -ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca 2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α 1A - and α 1B -ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

Recent advances in chemotherapeutics highlight the importance of molecularly-targeted perturbagens. Although these therapies typically address dysregulated cancer cell proteins, there are increasing therapeutic modalities that take into consideration cancer cell-extrinsic factors. Targeting components of tumor stroma such as vascular or immune cells has been shown to represent an efficacious approach in cancer treatment. Cancer-associated fibroblasts (CAFs) exemplify an important stromal component that can be exploited in targeted therapeutics, though their employment in drug discovery campaigns has been relatively minimal due to technical logistics in assaying for CAF-tumor interactions. Here we report a 3-dimensional multi-culture tumor:CAF spheroid phenotypic screening platform that can be applied to high-content drug discovery initiatives. Using a functional genomics approach we systematically profiled 1,024 candidate genes for CAF-intrinsic anti-spheroid activity; identifying several CAF genes important for development and maintenance of tumor:CAF co-culture spheroids. Along with previously reported genes such as WNT, we identify CAF-derived targets such as ARAF and COL3A1 upon which the tumor compartment depends for spheroid development. Specifically, we highlight the G-protein-coupled receptor OGR1 as a unique CAF-specific protein that may represent an attractive drug target for treating colorectal cancer. In vivo , murine colon tumor implants in OGR1 knockout mice displayed delayed tumor growth compared to tumors implanted in wild type littermate controls. These findings demonstrate a robust microphysiological screening approach for identifying new CAF targets that may be applied to drug discovery efforts., Competing Interests: CONFLICTS OF INTEREST None.