Recent advances in acid sensing by G protein coupled receptors.

Changes in extracellular proton concentrations occur in a variety of tissues over a range of timescales under physiological conditions and also accompany virtually all pathologies, notably cancers, stroke, inflammation and trauma. Proton-activated, G protein coupled receptors are already partially active at physiological extracellular proton concentrations and their activity increases with rising proton concentrations. Their ability to monitor and report changes in extracellular proton concentrations and hence extracellular pH appears to be involved in a variety of processes, and it is likely to mirror and in some cases promote disease progression. Unsurprisingly, therefore, these pH-sensing receptors (pHR) receive increasing attention from researchers working in an expanding range of research areas, from cellular neurophysiology to systemic inflammatory processes. This review is looking at progress made in the field of pHRs over the past few years and also highlights outstanding issues.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

Coincidence Detection of Membrane Stretch and Extracellular pH by the Proton-Sensing Receptor OGR1 (GPR68).

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.

Functional expression of calcium-permeable canonical transient receptor potential 4-containing channels promotes migration of medulloblastoma cells.

KEY POINTS: The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells. OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues. Activation of TRPC4-containing channels in DAOY cells, but not non-transformed granule precursor cells, results in prominent increases in [Ca2+ ]i and promotes cell motility in wound healing and transwell migration assays. Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca2+ ]i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4. Our results suggest that OGR1 enhances expression of TRPC4-containing channels that contribute to enhanced invasion and metastasis of granule precursor-derived human medulloblastoma. ABSTRACT: Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton-sensing Gq -coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton-potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed TRPC4. In DAOY cells, activation of TRPC4-containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration were observed. Our results suggest that OGR1-dependent increases in TRPC4 expression may favour formation of highly Ca2+ -permeable TRPC4-containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB.

Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H.

G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca(2+) and H(+), respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression.

Extracellular acidosis impairs P2Y receptor-mediated Ca(2+) signalling and migration of microglia.

Microglia are the resident macrophage and immune cell of the brain and are critically involved in combating disease and assaults on the brain. Virtually all brain pathologies are accompanied by acidosis of the interstitial fluid, meaning that microglia are exposed to an acidic environment. However, little is known about how extracellular acidosis impacts on microglial function. The activity of microglia is tightly controlled by 'on' and 'off' signals, the presence or absence of which results in generation of distinct phenotypes in microglia. Activation of G protein coupled purinergic (P2Y) receptors triggers a number of distinct behaviours in microglia, including activation, migration, and phagocytosis. Using pharmacological tools and fluorescence imaging of the murine cerebellar microglia cell line C8B4, we show that extracellular acidosis interferes with P2Y receptor-mediated Ca(2+) signalling in these cells. Distinct P2Y receptors give rise to signature intracellular Ca(2+) signals, and Ca(2+) release from stores and Ca(2+) influx are differentially affected by acidotic conditions: Ca(2+) release is virtually unaffected, whereas Ca(2+) influx, mediated at least in part by store-operated Ca(2+) channels, is profoundly inhibited. Furthermore, P2Y1 and P2Y6-mediated stimulation of migration is inhibited under conditions of extracellular acidosis, whereas basal migration independent of P2Y receptor activation is not. Taken together, our results demonstrate that an acidic microenvironment impacts on P2Y receptor-mediated Ca(2+) signalling, thereby influencing microglial responses and responsiveness to extracellular signals. This may result in altered behaviour of microglia under pathological conditions compared with microglial responses in healthy tissue.

Lack of kinase regulation of canonical transient receptor potential 3 (TRPC3) channel-dependent currents in cerebellar Purkinje cells.

Canonical transient receptor potential (TRPC) channels are widely expressed in the brain and play several roles in development and normal neuronal function. In the cerebellum, Purkinje cell TRPC3 channels underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation. In these cells TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long term depression (LTD), which underlies cerebellar motor learning. LTD induction requires protein kinase C (PKC) and protein kinase G (PKG) activation, and although PKC phosphorylation targets are well established, virtually nothing is known about PKG targets in LTD. Because TRPC3 channels are inhibited after phosphorylation by PKC and PKG in expression systems, we examined whether native TRPC3 channels in Purkinje cells are a target for PKG or PKC, thereby contributing to cerebellar LTD. We find that in Purkinje cells, activation of TRPC3-dependent currents is not inhibited by conventional PKC or PKG to any significant extent and that inhibition of these kinases does not significantly impact on TRPC3-mediated currents either. Based on these and previous findings, we propose that TRPC3-dependent currents may differ significantly in their regulation from those overexpressed in expression systems.

Activation of native TRPC3 cation channels by phospholipase D.

In the mammalian nervous system, stimulation of G-protein-coupled type I glutamate receptors triggers various forms of neuronal plasticity, including cerebellar long-term depression and hippocampal long-term potentiation. Activation of these receptors in the cerebellum also leads to a slow excitatory postsynaptic current mediated by nonselective TRPC3 cation channels. How TRPC3 channels are opened is unknown, although it is widely thought that channel gating requires phospholipase C activation. Using the patch-clamp technique and immunohistochemistry in rat cerebellar slices, we show that metabotropic glutamate receptors activate TRPC3 channels through the small GTP-binding protein Rho and subsequent phospholipase D stimulation. TRPC3 channel gating is independent of phospholipase C activity. These results reveal a new mechanism for the gating of the ubiquitous TRPC3 channel and identify a key role for phospholipase D in the generation of the slow excitatory postsynaptic current in cerebellar Purkinje cells.

A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice.

The hereditary ataxias are a complex group of neurological disorders characterized by the degeneration of the cerebellum and its associated connections. The molecular mechanisms that trigger the loss of Purkinje cells in this group of diseases remain incompletely understood. Here, we report a previously undescribed dominant mouse model of cerebellar ataxia, moonwalker (Mwk), that displays motor and coordination defects and loss of cerebellar Purkinje cells. Mwk mice harbor a gain-of-function mutation (T635A) in the Trpc3 gene encoding the nonselective transient receptor potential cation channel, type C3 (TRPC3), resulting in altered TRPC3 channel gating. TRPC3 is highly expressed in Purkinje cells during the phase of dendritogenesis. Interestingly, growth and differentiation of Purkinje cell dendritic arbors are profoundly impaired in Mwk mice. Our findings define a previously unknown role for TRPC3 in both dendritic development and survival of Purkinje cells, and provide a unique mechanism underlying cerebellar ataxia.

Differentiation impairs low pH-induced Ca2+ signaling and ERK phosphorylation in granule precursor tumour cells.

Extracellular acidification is a hallmark of a number of debilitating pathologies including cancer, ischemia and inflammation. We have recently shown that in human granule precursor tumour cells a fall in extracellular pH triggers increases in intracellular Ca(2+) concentration through activation of G-protein coupled proton-sensing receptors coupling to phospholipase C. This pH-dependent rise in cytosolic Ca(2+) led to activation of the extracellular signal-regulated kinase ERK, providing a mechanistic explanation of how extracellular acidification can promote tumour growth. We now find that differentiation of granule precursor tumour cells profoundly affects their ability to respond to extracellular acidification with gene transcription. Differentiating cells have a lower Ca(2+) release probability from intracellular Ca(2+) stores upon acidification and cells that respond have a significantly smaller and slower Ca(2+) signal than proliferating cells. Importantly, Ca(2+) release in differentiating cells fails to evoke ERK phosphorylation. This altered responsiveness of differentiating cells is not due to reduced proton-sensing receptor expression or diminished Ca(2+) store content. Rather, our results suggest that in differentiating cells, the proton-sensing receptor couples less effectively to phospholipase C activation and IP(3) formation. Hence, the ability of human granule cells to respond to extracellular acidification by generating Ca(2+) signals and ERK activation is state-dependent, being lost upon differentiation.

Extracellular acidification elicits spatially and temporally distinct Ca2+ signals.

Extracellular acidification accompanies neoplastic transformation of tissues and increases with tumor aggressiveness [1, 2]. The intracellular signaling cascade triggered by this process remains poorly understood and may be linked to recently discovered proton-activated G protein-coupled receptors such as OGR1 and G2A [3, 4]. Here, we report that OGR1 and G2A are expressed in human medulloblastoma tissue and its corresponding neuronal cell line. We show that extracellular acidification activates phospholipase C, IP(3) formation, and subsequent Ca2+ release from thapsigargin-sensitive stores in neurons. The number of responsive cells and the amount of Ca2+ released from stores correlated positively with the extent of extracellular acidification. Ca2+ release recruited the MEK/ERK pathway, providing a mechanistic explanation for how acidification stimulates cell growth. In addition, acidification activated Ca2+-permeable ion channels through a mechanism dependent on phospholipase C but independent of store depletion or a cytoplasmic Ca2+ rise. Hence, extracellular acidification, to levels seen in tumor tissue, activates temporally and spatially distinct pathways that elevate Ca2+ and may be directly relevant for tumor cell biology.

Spontaneous neurotransmitter release and Ca2+--how spontaneous is spontaneous neurotransmitter release?

Neurotransmitter release from neurons takes place at specialized structures called synapses. Action potential-evoked exocytosis requires Ca(2+) influx through voltage-gated Ca(2+) channels. Spontaneous vesicle fusion occurs both in the absence of action potentials and without any apparent stimulus and is hence thought to be Ca(2+)-independent. However, increasing evidence shows that this form of neurotransmitter discharge can be modulated by changes in intracellular Ca(2+) concentration, suggesting that it is not truly spontaneous. This idea is supported by the fact that spontaneous release can be modulated by interfering with proteins involved in the exocytotic process. Interestingly, modulation of spontaneous discharge at the level of the release machinery is not always accompanied by corresponding modulation of action potential-evoked release, suggesting that two independent processes may underlie spontaneous and action potential-evoked exocytosis, at least at some synapses. This provides an attractive model whereby cells can modulate the two forms of neurotransmitter liberation, which often serve different physiological roles, independently of each other.

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake.

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.

Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.