MLC1: A New Calcium-regulated Protein Conferring Calcium Dependence to Volume-regulated Anion Channels (VRAC) in Astrocytes.

Abstract MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

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Structural Insights into CB1 Receptor Biased Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms20081837 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1837 ◽

Cited By ~ 20

Author(s):

Al-Zoubi ◽

Morales ◽

Reggio

Keyword(s):

Signaling Pathways ◽

Conformational Changes ◽

Intracellular Signaling ◽

Cannabinoid Receptors ◽

Computational Modelling ◽

Structural Features ◽

Cb1 Receptor ◽

Effector Proteins ◽

Spectroscopic Techniques ◽

Receptor Ligands

The endocannabinoid system has emerged as a promising target for the treatment of numerous diseases, including cancer, neurodegenerative disorders, and metabolic syndromes. Thus far, two cannabinoid receptors, CB1 and CB2, have been discovered, which are found predominantly in the central nervous system (CB1) or the immune system (CB2), among other organs and tissues. CB1 receptor ligands have been shown to induce a complex pattern of intracellular effects. The binding of a ligand induces distinct conformational changes in the receptor, which will eventually translate into distinct intracellular signaling pathways through coupling to specific intracellular effector proteins. These proteins can mediate receptor desensitization, trafficking, or signaling. Ligand specificity and selectivity, complex cellular components, and the concomitant expression of other proteins (which either regulate the CB1 receptor or are regulated by the CB1 receptor) will affect the therapeutic outcome of its targeting. With an increased interest in G protein-coupled receptors (GPCR) research, in-depth studies using mutations, biological assays, and spectroscopic techniques (such as NMR, EPR, MS, FRET, and X-ray crystallography), as well as computational modelling, have begun to reveal a set of concerted structural features in Class A GPCRs which relate to signaling pathways and the mechanisms of ligand-induced activation, deactivation, or activity modulation. This review will focus on the structural features of the CB1 receptor, mutations known to bias its signaling, and reported studies of CB1 receptor ligands to control its specific signaling.

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Isoproterenol-induced beta-2 adrenergic receptor activation negatively regulates interleukin-2 signaling

Biochemical Journal ◽

10.1042/bcj20180503 ◽

2018 ◽

Vol 475 (18) ◽

pp. 2907-2923 ◽

Cited By ~ 2

Author(s):

Blanca E. Ruiz-Medina ◽

Denisse A. Cadena-Medina ◽

Edmundo Esparza ◽

Amy J. Arrieta ◽

Robert A. Kirken

Keyword(s):

Signaling Pathways ◽

Intracellular Signaling ◽

Adrenergic Receptor ◽

Interleukin 2 ◽

Chain Complex ◽

Receptor Activation ◽

Effector Proteins ◽

Cell Trafficking ◽

Gamma Chain ◽

Human Lymphoid Cell

Regulation of intracellular signaling pathways in lymphocytes is critical for cell homeostasis and immune response. Interleukin-2 (IL-2), a key regulator of lymphocytes, signals following receptor-ligand engagement and subsequent recruitment and activation of effector proteins including JAKs and STATs. Lymphocytes can also be regulated by the central nervous system through the β2 adrenergic receptor (β2AR) pathway which can affect cell trafficking, proliferation, differentiation, and cytokine production. The cross-talk between these two signaling pathways represents an important mechanism that has yet to be fully elucidated. The present study provides evidence for communication between the IL-2 receptor (IL-2R) and β2AR. Treatment of human lymphoid cell lines with the β2AR agonist isoproterenol (ISO) alone increased cAMP levels and mediated a stimulatory response by activating AKT and ERK to promote cell viability. Interestingly, ISO activation of β2AR also induced threonine phosphorylation of the IL-2Rβ. In contrast, ISO treatment prior to IL-2 stimulation produced an inhibitory signal that disrupted IL-2 induced activation of the JAK/STAT, MEK/ERK, and PI3K pathways by inhibiting the formation of the IL-2R beta–gamma chain complex, and subsequently cell proliferation. Moreover, γc-family cytokines-mediated STAT5 activation was also inhibited by ISO. These results suggest a molecular mechanism by which β2AR signaling can both stimulate and suppress lymphocyte responses and thus explain how certain therapeutic agents, such as vasodilators, may impact immune responsiveness.

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Superoxide Flux in Endothelial Cells via the Chloride Channel-3 Mediates Intracellular Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0830 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2002-2012 ◽

Cited By ~ 120

Author(s):

Brian J. Hawkins ◽

Muniswamy Madesh ◽

C. J. Kirkpatrick ◽

Aron B. Fisher

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Chloride Channel ◽

Intracellular Signaling ◽

Anion Channel ◽

Transmembrane Flux ◽

Cell Plasma ◽

Intracellular Ca ◽

Cellular Apoptosis ◽

Extracellular Side

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O2.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2.−, pulmonary microvascular endothelial cells were preloaded with the O2.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2.−resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O2.−triggered Ca2+release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2.−production and cellular apoptosis. These “signaling” effects of O2.−were prevented by DIDS treatment, by depletion of intracellular Ca2+stores with thapsigargin and by chelation of intracellular Ca2+. This study demonstrates that O2.−flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2+release, which activates mitochondrial O2.−generation.

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ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

AJP Cell Physiology ◽

10.1152/ajpcell.00330.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. C204-C213 ◽

Cited By ~ 84

Author(s):

Alexander A. Mongin ◽

Harold K. Kimelberg

Keyword(s):

Intracellular Signaling ◽

Anion Channel ◽

Cell Swelling ◽

Organic Osmolytes ◽

Signaling Mechanism ◽

Pharmacological Agents ◽

Organic Osmolyte ◽

Primary Astrocyte ◽

Intracellular Ca ◽

Rat Astrocytes

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl− currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on d-[3H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated d-[3H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (≥80% inhibition) by intracellular Ca2+ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control d-[3H]aspartate release activated by the substantial hyposmotic swelling showed little (≤25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca2+-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca2+/calmodulin-independent signaling mechanism. Ca2+-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication.

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Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.

The Journal of Cell Biology ◽

10.1083/jcb.133.5.997 ◽

1996 ◽

Vol 133 (5) ◽

pp. 997-1005 ◽

Cited By ~ 53

Author(s):

M H Cardone ◽

B L Smith ◽

P A Mennitt ◽

D Mochly-Rosen ◽

R B Silver ◽

...

Keyword(s):

Signaling Pathways ◽

Intracellular Signaling ◽

Membrane Traffic ◽

Polymeric Immunoglobulin Receptor ◽

Apical Surface ◽

Ligand Interaction ◽

Immunoglobulin Receptor ◽

Kinase C ◽

Intracellular Ca ◽

Second Messenger Pathways

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

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Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.1.c172 ◽

1994 ◽

Vol 266 (1) ◽

pp. C172-C178 ◽

Cited By ~ 72

Author(s):

H. Pasantes-Morales ◽

R. A. Murray ◽

R. Sanchez-Olea ◽

J. Moran

Keyword(s):

Amino Acids ◽

Regulatory Volume Decrease ◽

Anion Channel ◽

Inhibitory Effect ◽

Disulfonic Acid ◽

Channel Blockers ◽

Gamma Aminobutyric Acid ◽

Extracellular Concentration ◽

Cultured Astrocytes ◽

Volume Decrease

The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.

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