PIP2 Hydrolysis Research Articles

Live Cell Imaging and Optogenetics-Based Assays for GPCR Activity.

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gβγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.

Dissociation of the G protein βγ from the Gq–PLCβ complex partially attenuates PIP2 hydrolysis

Phospholipase C β (PLCβ), which is activated by the Gq family of heterotrimeric G proteins, hydrolyzes the inner membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). Because Gq and PLCβ regulate many crucial cellular processes and have been identified as major disease drivers, activation and termination of PLCβ signaling by the Gαq subunit have been extensively studied. Gq-coupled receptor activation induces intense and transient PIP2 hydrolysis, which subsequently recovers to a low-intensity steady-state equilibrium. However, the molecular underpinnings of this equilibrium remain unclear. Here, we explored the influence of signaling crosstalk between Gq and Gi/o pathways on PIP2 metabolism in living cells using single-cell and optogenetic approaches to spatially and temporally constrain signaling. Our data suggest that the Gβγ complex is a component of the highly efficient lipase GαqGTP–PLCβ–Gβγ. We found that over time, Gβγ dissociates from this lipase complex, leaving the less-efficient GαqGTP–PLCβ lipase complex and allowing the significant partial recovery of PIP2 levels. Our findings also indicate that the subtype of the Gγ subunit in Gβγ fine-tunes the lipase activity of Gq–PLCβ, in which cells expressing Gγ with higher plasma membrane interaction show lower PIP2 recovery. Given that Gγ shows cell- and tissue-specific subtype expression, our findings suggest the existence of tissue-specific distinct Gq–PLCβ signaling paradigms. Furthermore, these results also outline a molecular process that likely safeguards cells from excessive Gq signaling.

A metabolic reaction-diffusion model for PKCα translocation via PIP2 hydrolysis in an endothelial cell.

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) at the cell membrane induces the release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm and diffusion of diacylglycerol (DAG) through the membrane, respectively. Release of IP3 subsequently increases Ca2+ levels in the cytoplasm, which results in activation of protein kinase C α (PKCα) by Ca2+ and DAG, and finally the translocation of PKCα from the cytoplasm to the membrane. In this study, we developed a metabolic reaction-diffusion framework to simulate PKCα translocation via PIP2 hydrolysis in an endothelial cell. A three-dimensional cell model, divided into membrane and cytoplasm domains, was reconstructed from confocal microscopy images. The associated metabolic reactions were divided into their corresponding domain; PIP2 hydrolysis at the membrane domain resulted in DAG diffusion at the membrane domain and IP3 release into the cytoplasm domain. In the cytoplasm domain, Ca2+ was released from the endoplasmic reticulum, and IP3, Ca2+, and PKCα diffused through the cytoplasm. PKCα bound Ca2+ at, and diffused through, the cytoplasm, and was finally activated by binding with DAG at the membrane. Using our model, we analyzed IP3 and DAG dynamics, Ca2+ waves, and PKCα translocation in response to a microscopic stimulus. We found a qualitative agreement between our simulation results and our experimental results obtained by live-cell imaging. Interestingly, our results suggest that PKCα translocation is dominated by DAG dynamics. This three-dimensional reaction-diffusion mathematical framework could be used to investigate the link between PKCα activation in a cell and cell function.

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane.

Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles – as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

PIP2: A critical regulator of vascular ion channels hiding in plain sight

The phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), has long been established as a major contributor to intracellular signaling, primarily by virtue of its role as a substrate for phospholipase C (PLC). Signaling by Gq-protein-coupled receptors triggers PLC-mediated hydrolysis of PIP2 into inositol 1,4,5-trisphosphate and diacylglycerol, which are well known to modulate vascular ion channel activity. Often overlooked, however, is the role PIP2 itself plays in this regulation. Although numerous reports have demonstrated that PIP2 is critical for ion channel regulation, how it impacts vascular function has received scant attention. In this review, we focus on PIP2 as a regulator of ion channels in smooth muscle cells and endothelial cells-the two major classes of vascular cells. We further address the concerted effects of such regulation on vascular function and blood flow control. We close with a consideration of current knowledge regarding disruption of PIP2 regulation of vascular ion channels in disease.

Diacylglycerol kinases regulate TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is activated by heat and by capsaicin, the pungent compound in chili peppers. Calcium influx through TRPV1 has been shown to activate a calcium-sensitive phospholipase C (PLC) enzyme and to lead to a robust decrease in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels, which is a major contributor to channel desensitization. Diacylglycerol (DAG), the product of the PLC-catalyzed PI(4,5)P2 hydrolysis, activates protein kinase C (PKC). PKC is known to potentiate TRPV1 activity during activation of G protein-coupled receptors, but it is not known whether DAG modulates TRPV1 during desensitization. We found here that inhibition of diacylglycerol kinase (DAGK) enzymes reduces desensitization of native TRPV1 in dorsal root ganglion neurons as well as of recombinant TRPV1 expressed in HEK293 cells. The effect of DAGK inhibition was eliminated by mutating two PKC-targeted phosphorylation sites, Ser-502 and Ser-800, indicating involvement of PKC. TRPV1 activation induced only a small and transient increase in DAG levels, unlike the robust and more sustained increase induced by muscarinic receptor activation. DAGK inhibition substantially increased the DAG signal evoked by TRPV1 activation but not that evoked by M1 muscarinic receptor activation. Our results show that Ca2+ influx through TRPV1 activates PLC and DAGK enzymes and that the latter limits formation of DAG and negatively regulates TRPV1 channel activity. Our findings uncover a role of DAGK in ion channel regulation.

Intracellular acidification facilitates receptor-operated TRPC4 activation through PLCδ1 in a Ca2+ -dependent manner.

Receptor-operated activation of TRPC4 cation channels requires Gi/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca2+ . Concurrent stimulation of the Gq/11 pathway accelerates Gi/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca2+ . The kinetic effect of Gq/11 was diminished by alkaline intracellular pH (pHi ) and increased pHi buffer capacity. Acidic pHi (6.75-6.25) together with the cytosolic Ca2+ rise accelerated Gi/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca2+ -dependent activation of PLCδ1. The data suggest that the Gq/11 -PLCβ pathway facilitates Gi/o activation of TRPC4 through hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2 ) to produce the initial proton signal that triggers a self-propagating PLCδ1 activity supported by regenerative H+ and Ca2+ . The findings provide novel mechanistic insights into receptor-operated TRPC4 activation by coincident Gq/11 and Gi/o pathways and shed light on how aberrant activation of TRPC4 may occur under pathological conditions to cause cell damage. Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels activated downstream from receptors that signal through G proteins. Our recent work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The Gi/o -mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ), the substrate hydrolysed by PLC, and intracellular Ca2+ . As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels remains obscure. Here, using patch-clamp recordings of HEK293 cells heterologously co-expressing mouse TRPC4β and the Gi/o -coupled μ opioid receptor, we investigated the role of intracellular protons on Gi/o -mediated TRPC4 activation. We found that acidic cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation by directly acting upon TRPC4. We additionally demonstrated that protons exert their effect through sensitization of PLCδ1 to Ca2+ , which in turn promotes PLCδ1 activity and further potentiates TRPC4 via a positive feedback mechanism. The mechanism elucidated here helps explain how Gi/o and Gq/11 co-stimulation induces a faster activation of TRPC4 than Gi/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.

The phosphatidinositol-transfer protein Nir3 is a modulator of T cell development

Abstract Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) functions as a critical source of second messengers implicated in T cell activation. In T cells, hydrolysis of PIP2 by the lipid phosphatase PLCγ1 generates the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), resulting in PKC and Ras/MAPK activation as well as calcium increases that contribute to T cell responses. A secondary consequence of PLCγ1 activation is a transient reduction in PIP2 levels. The PI transfer proteins Nir2 and Nir3 have been shown to replenish PIP2 levels at the PM through delivery of the precursor PI from the endoplasmic reticulum (ER) via non-vesicular transport. In particular, Nir3 has been shown to replenish PI levels during basal signaling and in response to weak signaling. Given the importance of weak signaling in thymocyte positive selection, as well as the elevated levels of Nir3 mRNA during the double positive (DP) stage of thymocytes development (Immgen), we hypothesize that Nir3 is a regulator of PI homeostasis during thymocyte development. Using CRISPR-Cas9 technology, we have generated a Nir3 knockout mouse. DP thymocytes from Nir3 knockout mice have reduced basal calcium levels, as well as reduced TCR-induced calcium flux. Furthermore, introduction of the OT1 transgenic TCR into Nir3 knockout mice resulted in elevated numbers of DP thymocytes and reduced numbers of CD8SP thymocytes, suggesting a block in positive selection. Analysis of TCR signaling in Nir3 knockout OT1 thymocytes revealed attenuated calcium flux as well as defective induction of pERK, pAkt, and pS6. These findings are the first to characterize the function of PI transfer proteins and their importance specifically in T cell development.

Dynamics of allosteric activation in PLC‐γ isozymes

The PLC‐γ isozymes (‐γ1, ‐γ2) hydrolyze the minor membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) into the second messengers diacylglycerol and inositol 1,4,5‐trisphosphate to control diverse biological processes including chemotaxis, vascular development, and immune function. In contrast, mutant forms of the PLC‐γ isozymes that are constitutively active promote autoimmune disorders and cancer. For example, PLC‐γ1 is the most frequently (~40%) mutated gene in patients with adult T‐cell leukemia/lymphoma. However, despite widespread appreciation of the PLC‐γ isozymes in diverse biological processes, their regulation remains poorly understood.We recently determined the crystal structure of full‐length PLC‐γ1 at high resolution. The structure highlights a conserved catalytic core blocked from accessing membranes by a regulatory array unique to the PLC‐γ isozymes. Within the regulatory array, an N‐terminal SH2 (nSH2) domain is arranged to engage phosphorylated portions of tyrosine kinases. Complex formation facilitates the recruitment of PLC‐γ1 to membranes and the phosphorylation of Tyr783, also within the regulatory array. In contrast to the nSH2 domain, the equivalent surface of the C‐terminal SH2 (cSH2) domain is blocked by interactions with the catalytic core. Nonetheless, this surface must bind phosphorylated Tyr783 for lipase activation.We hypothesize that engagement of a kinase to the nSH2 domain propagates structural alterations into the cSH2 domain that destabilize its interactions with the catalytic core thus priming it to bind phosphorylated Tyr783. This interaction presumably leads to a larger rearrangement of the regulatory array relative to the catalytic core necessary for membrane engagement and PIP2 hydrolysis. This hypothesis is supported by alterations highlighted in the structural comparison of full‐length PLC‐γ1 with a pre‐existing structure of the two SH2 domains bound to the kinase domain of fibroblast growth factor receptor 1 (FGFR1K). To test this hypothesis, we used a fluorogenic PIP2 analog and hydrogen‐deuterium exchange coupled to mass spectrometry to assess activity and structural changes in PLC‐γ1 upon engagement of FGFR1K, respectively.Here we show that binding of FGFR1K to PLC‐γ1 increases deuterium incorporation within the interface between the regulatory array and the catalytic core suggesting that interaction of FGFR1K and PLC‐γ1 disrupts autoinhibition. The addition of liposomes further increases deuterium incorporation within the same region suggesting that membranes reinforce this disruption. Similar, but more dramatic increases in deuterium incorporation were observed for the cancer‐associated and constitutively active variant, PLC‐γ1 (D1165H). Finally, we show that a two‐fold molar excess of FGFR1K relative to PLC‐γ1 increased lipase activity up to three‐fold. Taken together, these data strongly suggest that kinases shift the conformational equilibrium of PLC‐γ isozymes to facilitate access and hydrolysis of membrane‐resident PIP2. This allosteric effect is recapitulated by oncogenic mutations and reinforced by membranes.Support or Funding InformationThis work was supported by NIH Grant R01‐GM057391 (J.S.). E.S‐P. was supported by an NSF GRF under Grant DGE‐1650116.

Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1

Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.Support or Funding InformationThis research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.

OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis.

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation.

TRPC Channels in the SOCE Scenario

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario.

Phosphoinositide signaling and regulation in Trypanosoma brucei: Specialized functions in a protozoan pathogen.

Trypanosoma brucei is a single-celled protozoan pathogen that causes human and animal trypanosomiasis and incurs devastating health and economic burdens in Africa. Together with the related parasites T. cruzi and Leishmania spp., which cause Chagas disease and leishmaniasis, respectively, over 8 million people are affected annually worldwide [1]. These parasites alternate between a mammalian host and the insect vector and undergo extensive developmental changes during their life cycle, including changes in surface coat, gene expression, metabolism, and organelle morphology and function. They also have elaborate mechanisms of gene regulation that control the expression of genes involved in host immune evasion during infection. The control of developmental changes and immune evasion mechanisms entails a complex network of signaling and regulatory processes that includes phosphatidylinositol (PI) phosphates (PIP, also called phosphoinositides) and inositol phosphates (IP) [2–8]. PIPs and IPs are ubiquitous in eukaryotes and consist of a subset of molecules containing mono or poly phosphorylated inositol (Fig 1A). Whilst PIPs are a class of phospholipids generally associated with cellular or organellar membranes and produced via phosphorylation of PI, IPs are soluble molecules produced as a result of PIP hydrolysis by phospholipase enzymes. PIPs and IPs interact with proteins or RNA and regulate numerous cellular functions in eukaryotes. As detailed below, these metabolites and related enzymes function as a regulatory system with essential roles in T. brucei metabolism and development [6], trafficking and organelle biogenesis [9–11], Ca2+ signaling [12], and immune evasion mechanisms [5, 7]. Open in a separate window Fig 1 PIP and IP synthesis and regulation in T. brucei. (A) Structure of PIP2 indicated by the inositol ring (black hexagon), phosphates (red circles), and DAG with fatty acid chain. PLC cleaves PIP2 and produces diacylglycerol and IP3. Black arrows indicate phosphate and inositol. The yellow arrow indicates the site of PLC cleavage, which occurs between DAG and phosphate sn1. The green arrow indicates the directionality of the PLC reaction. (B) The number of genes involved in PIP and IP synthesis, signaling (includes PLC and IP3 receptors), and PIP and IP kinases and phosphatases in eukaryotes and prokaryotes. The size of the black circles indicates the number of genes in each category. (C) Synthesis of PIPs and IPs based on T. brucei predicted and characterized enzymes. Enzymes, whose regulatory functions are discussed here, are indicated in blue. PIP-Pase indicates enzymes that dephosphorylate PIPs at positions 3, 4, or 5 of the inositol ring. It includes PIP5Pase, whose catalytic activity is detailed below in D. Metabolite short names are used for simplicity. (D) Regulation of VSG silencing by PIP5Pase. PIP5Pase dephosphorylates the 5-phosphate (green circle) of PIP3 and prevents this metabolite binding to RAP1, which preserves RAP1 function (and likely other proteins) in ES chromatin organization. Catalytic inactivation of PIP5Pase results in PIP3 binding to RAP1, which affects ES chromatin organization and results in transcription of VSG genes. 1, diacylglycerol kinase; 2, cytidine diphosphate-diacylglycerol synthase; 3, phosphatidylinositol synthase; 70 bp, 70 base pair repeats; Ath, Arabidopsis thaliana; DAG, diacylglycerol; ER, endoplasmic reticulum; ES, expression site; ESAG, expression site associated genes; Hsp, Homo sapiens; I, myo-inositol; IMPase, inositol monophosphatase; IP, inositol phosphate; IP1, D-myo-inositol 1-monophosphate; IP2, D-myo-inositol 1,4-diphosphate; IP3, D-myo-inositol 1,4,5-triphosphate; IP4, D-myo-inositol 1,3,4,5-tetrakisphosphate; IP5, D-myo-inositol 1,2,3,4,5-pentakisphosphate; IP5Pase, inositol polyphosphate 5-phosphatase; IP6, D-myo-inositol 1,2,3,4,5,6-hexakisphosphate; IP6K, inositol hexakisphosphate kinase; IP7, D-myo-inositol 5-diphospho 1,2,3,4,6-pentakisphosphate; IPMK, inositol polyphosphate multikinase; Mtb, Mycobacterium tuberculosis; PIP, phosphatidylinositol phosphate; PIP1, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-biphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PIP5K, phosphatidylinositol phosphate 5-kinase; PIP5Pase, phosphatidylinositol phosphate 5-phosphatase; PIP-Pase, phosphatidylinositol phosphate phosphatases; PLC, Phospholipase C; PM, plasma membrane; Pol I, RNA polymerase I; PP-IP4, D-myo-inositol 5-diphospho 1,3,4,6-tetrakisphosphate; RAP1, repressor-activator protein 1; Sce, Saccharomyces cerevisiae; sn1, unimolecular nucleophilic substitution; Tbr, T. brucei; Ttm, Thermus thermophilus; VSG, variant surface glycoprotein.

ORP1L, ORP1S, and ORP2: Lipid Sensors and Transporters

Lipid transfer proteins are crucial for intracellular cholesterol trafficking at sites of membrane contact. In the OSBP/ORPs (oxysterol binding protein and OSBP-related proteins) family of lipid transfer proteins, ORP1L, ORP1S and ORP2 play important roles in cholesterol transport. ORP1L is an endosome/lysosome-anchored cholesterol sensor which may also move cholesterol bidirectionally at the interface between the endoplasmic reticulum and the endosome/lysosome. ORP2 delivers cholesterol to the plasma membrane, driven by PI(4,5)P2hydrolysis. ORP1S may also transport cholesterol to the plasma membrane, although it is unclear if phosphoinositides are involved. The source of cholesterol delivered to the plasma membrane by ORP1S and ORP2 remains unclear. This review summarises the roles of these proteins in maintaining cellular cholesterol homeostasis and in human disease.

Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4)

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Mal protein stabilizes luminal membrane PLC-β3 and negatively regulates ENaC in mouse cortical collecting duct cells.

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl- cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

ENHANCED FUNCTION OF PLCG2 VARIANT P522R IN MACROPHAGES AND MICROGLIA IS ASSOCIATED WITH PROTECTION FROM ALZHEIMER’S DISEASE

Evidence from GWAS suggests the P522R variant in the enzyme PLCg2 is protective against Alzheimer's disease (AD). This variant lies in the auto-inhibitory component of the PLCg2 gene, and such mutations could possibly alter the activation of PLCg2. Expression of PLCg2 in the brain is predominantly found in microglia and microglia have been implicated in the risk of AD. PLCg2 catalyses the hydrolysis of PIP2 to IP3 and DAG, with IP3 causing intracellular Ca2+ release. Intracellular Ca2+ release causes multiple cellular responses, such as phagocytosis. This suggests that the P522R variant may alter the responsiveness of microglia in conditions such as AD. Several techniques were utilized to investigate function of PLCg2 within the variant. This included molecular dynamic stimulation (GROMACS), run in the NPT ensemble and resulting structures were analysed using RMSD and visualized for structural differences using VMD. In addition, we investigated cytoplasmic Ca2+ increase (Fura2 and Fluo-8), indicative of a signalling response, following activation of PLCg2 in human (using anti-CD32) and murine (using anti-CD16/32 and Ab) models. Novel human P522R KOLF2 iPSC lines and P522R knockin mice were generated using CRISPR-assisted gene-targeting. We used iPSC-derived human microglia and primary murine cortical and hippocampal microglia alongside macrophages derived from conditionally-immortalized mouse macrophage precursor cell lines. The requirement for PLCg2 in the intracellular Ca2+ changes were investigated using specific PLCg inhibitors (edelfosine, U73122) and a specific GapmeR knockdown system. Computational models suggest that structural changes in the P522R mutant would reduce binding of inhibitors. In addition, Ca2+ signalling assays in all the cellular models consistently demonstrated increased cytosolic Ca2+ release in P522R variants compared to controls following activation of PLCg2. Our results are the first to show that the protective P522R variant exhibits increased activity in microglia and macrophages in response to physiological stimuli. This protection most likely results from alteration of microglia response after exposure to the environmental cues present in AD. PLCg2 represents a strong pharmacological target suggesting the possibility of a new intervention site for AD.

Gαq Sensitizes TRPM8 to Inhibition by PI(4,5)P2 Depletion upon Receptor Activation.

The cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein-coupled receptors (GPCRs) by proinflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of Gαq to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] for activity. However, it was claimed that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here, we show that supplementing the whole-cell patch pipette with PI(4,5)P2 reduced inhibition of TRPM8 by activation of Gαq-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated phospholipase C (PLC) and decreased plasma membrane PI(4,5)P2 levels in these neurons. PI(4,5)P2 also reduced inhibition of TRPM8 by activation of heterologously expressed muscarinic M1 receptors. Coexpression of a constitutively active Gαq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein, decreasing PI(4,5)P2 levels using a voltage-sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that, upon GPCR activation, Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P2 and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P2 levels.SIGNIFICANCE STATEMENT Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) ion channels. Most inflammatory mediators act via G-protein-coupled receptors that activate the phospholipase C pathway, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here, we propose that direct binding of Gαq both reduces TRPM8 activity and sensitizes the channel to inhibition by decreased levels of its cofactor, PI(4,5)P2 Our data demonstrate the convergence of two downstream effectors of receptor activation, Gαq and PI(4,5)P2 hydrolysis, in the regulation of TRPM8.

Amyloid \u03b2 oligomers suppress excitatory transmitter release via presynaptic depletion of phosphatidylinositol-4,5-bisphosphate

Amyloid β (Aβ) oligomer-induced aberrant neurotransmitter release is proposed to be a crucial early event leading to synapse dysfunction in Alzheimer’s disease (AD). In the present study, we report that the release probability (Pr) at the synapse between the Schaffer collateral (SC) and CA1 pyramidal neurons is significantly reduced at an early stage in mouse models of AD with elevated Aβ production. High nanomolar synthetic oligomeric Aβ42 also suppresses Pr at the SC-CA1 synapse in wild-type mice. This Aβ-induced suppression of Pr is mainly due to an mGluR5-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) in axons. Selectively inhibiting Aβ-induced PIP2 hydrolysis in the CA3 region of the hippocampus strongly prevents oligomeric Aβ-induced suppression of Pr at the SC-CA1 synapse and rescues synaptic and spatial learning and memory deficits in APP/PS1 mice. These results first reveal the presynaptic mGluR5-PIP2 pathway whereby oligomeric Aβ induces early synaptic deficits in AD.

G protein αq exerts expression level-dependent distinct signaling paradigms

G protein αq-coupled receptors (Gq-GPCRs) primarily signal through GαqGTP mediated phospholipase Cβ (PLCβ) stimulation and the subsequent hydrolysis of phosphatidylinositol 4, 5 bisphosphate (PIP2). Though Gq-heterotrimer activation results in both GαqGTP and Gβγ, unlike Gi/o-receptors, it is unclear if Gq-coupled receptors employ Gβγ as a major signal transducer. Compared to Gi/o- and Gs-coupled receptors, we observed that most cell types exhibit a limited free Gβγ generation upon Gq-pathway and Gαq/11 heterotrimer activation. We show that cells transfected with Gαq or endogenously expressing more than average-levels of Gαq/11 compared to Gαs and Gαi exhibit a distinct signaling regime primarily characterized by recovery-resistant PIP2 hydrolysis. Interestingly, the elevated Gq-expression is also associated with enhanced free Gβγ generation and signaling. Furthermore, the gene GNAQ, which encodes for Gαq, has recently been identified as a cancer driver gene. We also show that GNAQ is overexpressed in tumor samples of patients with Kidney Chromophobe (KICH) and Kidney renal papillary (KIRP) cell carcinomas in a matched tumor-normal sample analysis, which demonstrates the clinical significance of Gαq expression. Overall, our data indicates that cells usually express low Gαq levels, likely safeguarding cells from excessive calcium as wells as from Gβγ signaling.