GPR68 limits the severity of chemical-induced oral epithelial dysplasia.

Head and neck cancer is the sixth most common malignancy, and there is an urgent need to identify physiological processes contributing to tumorigenesis. Extracellular acidification caused by aerobic glycolysis within tumor microenvironments can stimulate proton-sensing receptors. GPR68, or ovarian cancer G protein-coupled receptor 1, responds to extracellular acidity and is highly expressed in head and neck squamous cell carcinoma (HNSCC) as well as normal esophageal tissue. To study the role of GPR68 in oral dysplasia, wild-type and GPR68-/- mice were treated with 4-Nitroquinoline N-oxide (4NQO) in drinking water for 11-13 weeks, followed by normal water for 11-12 weeks. 4NQO treatment resulted in 45 percent of GPR68-/- mice developing severe dysplasia or squamous cell carcinoma compared to only 10.5 percent of GPR68+/+ mice. This correlated with increased frequencies of regulatory T cells in the spleens of male GPR68-/- mice. Dysplastic regions of the tongue had increased CD31 staining compared to normal regions in both GPR68-/- and GPR68+/+ mice, suggesting that angiogenesis was GPR68-independent. RNA knockdown studies using HNSCC cell lines demonstrated no direct effect of GPR68 on survival or growth. Overall, we demonstrate that GPR68-deficiency worsens the severity of chemical-induced oral dysplasia, suggesting a protective role for this gene in tumorigenesis.

pH and proton-sensitive receptors in brain ischemia.

Extracellular proton concentration is at 40 nM when pH is 7.4. In disease conditions such as brain ischemia, proton concentration can reach µM range. To respond to this increase in extracellular proton concentration, the mammalian brain expresses at least three classes of proton receptors. Acid-sensing ion channels (ASICs) are the main neuronal cationic proton receptor. The proton-activated chloride channel (PAC), which is also known as (aka) acid-sensitive outwardly rectifying anion channel (ASOR; TMEM206), mediates acid-induced chloride currents. Besides proton-activated channels, GPR4, GPR65 (aka TDAG8, T-cell death-associated gene 8), and GPR68 (aka OGR1, ovarian cancer G protein-coupled receptor 1) function as proton-sensitive G protein-coupled receptors (GPCRs). Though earlier studies on these GPCRs mainly focus on peripheral cells, we and others have recently provided evidence for their functional importance in brain injury. Specifically, GPR4 shows strong expression in brain endothelium, GPR65 is present in a fraction of microglia, while GPR68 exhibits predominant expression in brain neurons. Here, to get a better view of brain acid signaling and its contribution to ischemic injury, we will review the recent findings regarding the differential contribution of proton-sensitive GPCRs to cerebrovascular function, neuroinflammation, and neuronal injury following acidosis and brain ischemia.

G protein-coupled receptor 68 increases the number of B lymphocytes.

G protein-coupled receptor 68 (GPR68) is a proton sensor that is activated upon binding to extracellular protons. We have previously found that GPR68 induces a proapoptotic pathway in bone marrow (BM) cells from the patients with myelodysplastic syndromes (MDS) after treated with lenalidomide. However, the function of GPR68 in normal hematopoietic cells remains unclear. With genetic loss of function approach, we found reduced frequency and number of B lymphocytes in the peripheral blood (PB) of whole body Gpr68-/- mice compared to control littermates upon aging. During hematopoietic regeneration, such as in response to fluorouracil (5-FU), we also found reduced frequency and number of B lymphocytes in Gpr68-/- mice compared to wild type mice. Mechanism studies revealed that Gpr68 expression was upregulated in B lymphocytes of BM during aging and in hematopoietic progenitor cells after treatment with 5-FU. In addition, activation of Gpr68 by its activators increased the frequency and number of B lymphocytes. Our studies indicate that Gpr68 expression is upregulated in hematopoietic cells upon aging and during hematopoietic regeneration that ends up with increased number of B lymphocytes.

Acid-Sensing Ion Channels Contribute to Type III Adenylyl Cyclase-Independent Acid Sensing of Mouse Olfactory Sensory Neurons.

Acids can disturb the ecosystem of wild animals through altering their olfaction and olfaction-related survival behaviors. It is known that the main olfactory epithelia (MOE) of mammals rely on odorant receptors and type III adenylyl cyclase (AC3) to detect general odorants. However, it is unknown how the olfactory system sense protons or acidic odorants. Here, we show that while the MOE of AC3 knockout (KO) mice failed to respond to an odor mix in electro-olfactogram (EOG) recordings, it retained a small fraction of acid-evoked EOG responses. The acetic acid-induced EOG responses in wild-type (WT) MOE can be dissected into two components: the big component dependent on the AC3-mediated cAMP pathway and the much smaller component not. The small acid-evoked EOG response of the AC3 KOs was blocked by diminazene, an inhibitor of acid-sensing ion channels (ASICs), but not by forskolin/IBMX that desensitize the cAMP pathway. AC3 KO mice lost their sensitivity to detect pungent odorants but maintained sniffing behavior to acetic acid. Immunofluorescence staining demonstrated that ASIC1 proteins were highly expressed in olfactory sensory neurons (OSNs), mostly enriched in the knobs, dendrites, and somata, but not in olfactory cilia. Real-time polymerase chain reaction further detected the mRNA expression of ASIC1a, ASIC2b, and ASIC3 in the MOE. Additionally, mice exhibited reduced preference to attractive objects when placed in an environment with acidic volatiles. Together, we conclude that the mouse olfactory system has a non-conventional, likely ASIC-mediated ionotropic mechanism for acid sensing.

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala.

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na(+)- and Ca(2+)-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.

Inhibition of human acid-sensing ion channel 1b by zinc.

Acid-sensing ion channel 1b (ASIC1b) is expressed in peripheral sensory neurons and has been implicated in nociception. Understanding the modulation of ASIC1b will provide important insight into how ASIC1b contributes to pain sensation. In our previous study, we showed that zinc, an important modulator of pain sensation, reduces rat ASIC1b current. However, rat ASIC1b shows several important differences from its recently identified human homolog. Most noticeably, human ASIC1b (hASIC1b) has a sustained component, which may play a role in persistent pain. Therefore, we tested here the hypothesis that zinc modulates the current properties of hASIC1b. Bath application of zinc suppressed the peak amplitude of hASIC1b currents, with a half-maximum inhibitory concentration of 37 µM. However, zinc did not affect the sustained component of hASIC1b currents. The effect of zinc was independent of pH-dependent activation, steady-state desensitization, and extracellular Ca(2+), suggesting noncompetitive mechanisms. Further, we found that extracellular site(s) of the hASIC1b subunit is important for the effect of zinc. Mutating cysteine 196, but not cysteine 309, in the extracellular domain of the hASIC1b abolished the zinc inhibition. These results suggest that, through modulating cysteine196, zinc may have a modulatory role in acute pain.

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density.

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased. Amplitude and decay time of mEPSCs, decay time of mIPSCs, and spine size were unaltered. Mice in which the PKA anchoring C-terminal 36 residues of AKAP150 are deleted (D36) showed similar changes. Furthermore, whereas acute stimulation of PKA (2-4 h) increases spine density, prolonged PKA stimulation (48 h) reduces spine density in apical dendrites of CA1 pyramidal neurons in organotypic slice cultures. The data from the AKAP150 mutant mice show that AKAP150-anchored PKA chronically limits the number of spines with functional AMPARs at 2-4 weeks of age. However, synaptic transmission and spine density was normal at 8 weeks in KO and D36 mice. Thus AKAP150-independent mechanisms correct the aberrantly high number of active spines in juvenile AKAP150 KO and D36 mice during development.

Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits.

The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H(2)O(2) increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H(2)O(2)-induced decrease in H(+)-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges.

Targeting ASIC1a reduces innate fear and alters neuronal activity in the fear circuit.

BACKGROUND: The molecular mechanisms underlying innate fear are poorly understood. Previous studies indicated that the acid sensing ion channel ASIC1a influences fear behavior in conditioning paradigms. However, these differences may have resulted from an ASIC1a effect on learning, memory, or the expression of fear. METHODS: To test the hypothesis that ASIC1a influences the expression of fear or anxiety independent of classical conditioning, we examined the effects of disrupting the mouse ASIC1a gene on unconditioned fear in the open field test, unconditioned acoustic startle, and fear evoked by the predator odor trimethylthiazoline (TMT). In addition, we tested the effects of acutely inhibiting ASIC1a with PcTx, an ASIC1a antagonist in tarantula venom. Our immunohistochemistry suggested ASIC1a is expressed in the bed nucleus of the stria terminalis, medial amygdala, and periaqueductal gray, which are thought to play important roles in the generation and expression of innate fear. Therefore, we also tested whether ASIC1a disruption altered c-fos expression in these structures following TMT exposure. RESULTS: We found that the loss of ASIC1a reduced fear in the open field test, reduced acoustic startle, and inhibited the fear response to TMT. Similarly, intracerebroventricular administration of PcTx reduced TMT-evoked freezing in ASIC1a(+/+) mice but not ASIC1a(-/-) mice. In addition, loss of ASIC1a altered TMT-evoked c-fos expression in the medial amydala and dorsal periaqueductal gray. CONCLUSIONS: These findings suggest that ASIC1a modulates activity in the circuits underlying innate fear. Furthermore, the data indicate that targeting the ASIC1a gene or acutely inhibiting ASIC1a suppresses fear and anxiety independent of conditioning.

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines.

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca(2+)-mediated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca(2+) concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca(2+) concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

An extranuclear locus of cAMP-dependent protein kinase action is necessary and sufficient for promotion of spiral ganglion neuronal survival by cAMP.

We showed previously that cAMP is a survival-promoting stimulus for cultured postnatal rat spiral ganglion neurons (SGNs) and that depolarization promotes SGN survival in part via recruitment of cAMP signaling. We here investigate the subcellular locus of cAMP prosurvival signaling. Transfection of GPKI, a green fluorescent protein (GFP)-tagged cAMP-dependent protein kinase (PKA) inhibitor, inhibits the ability of the permeant cAMP analog cpt-cAMP [8-(4-chlorophenylthio)-cAMP] to promote survival, indicating that PKA activity is necessary. Transfection of GFP-tagged PKA (GPKA) is sufficient to promote SGN survival, but restriction of GPKA to the nucleus by addition of a nuclear localization signal (GPKAnls) almost completely abrogates its prosurvival effect. In contrast, GPKA targeted to the extranuclear cytoplasm by addition of a nuclear export signal (GPKAnes) promotes SGN survival as effectively as does GPKA. Moreover, GPKI targeted to the nucleus lacks inhibitory effect on SGN survival attributable to cpt-cAMP or depolarization. These data indicate an extranuclear target of PKA for promotion of neuronal survival. Consistent with this, we find that dominant-inhibitory CREB mutants inhibit the prosurvival effect of depolarization but not that of cpt-cAMP. SGN survival is compromised by overexpression of the proapoptotic regulator Bad, previously shown to be phosphorylated in the cytoplasm by PKA. This Bad-induced apoptosis is prevented by cpt-cAMP or by cotransfection of GPKA or of GPKAnes but not of GPKAnls. Thus, cAMP prevents SGN death through a cytoplasmic as opposed to nuclear action, and inactivation of Bad proapoptotic function is a mechanism by which PKA can prevent neuronal death.