Membrane channels as integrators of G-protein-mediated signaling

A variety of extracellular stimuli regulate cellular responses via membrane receptors. A well-known group of seven-transmembrane domain-containing proteins referred to as G protein-coupled receptors, directly couple with the intracellular GTP-binding proteins (G proteins) across cell membranes and trigger various cellular responses by regulating the activity of several enzymes as well as ion channels. Many specific populations of ion channels are directly controlled by G proteins; however, indirect modulation of some channels by G protein-dependent phosphorylation events and lipid metabolism is also observed. G protein-mediated diverse modifications affect the ion channel activities and spatio-temporally regulate membrane potentials as well as of intracellular Ca^2 + concentrations in both excitatory and non-excitatory cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

A role for lipid rafts in immune cell signaling

Cross-linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src-family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.     

The region-specific activities of lipid rafts during axon growth and guidance

The generation and control of cell polarity is a fundamental mechanism for directed migration of the cell. In developing neurons, the axonal growth cone recognizes environmental molecular cues and migrates toward its correct target, thereby forming neuronal networks. The spatial information provided by environmental cues directs axon growth and guidance through generating polarity of intracellular signals and cytoskeletal organization in the growth cone. This polarization process is dependent on lipid rafts, specialized microdomains in the cell membrane. Lipid rafts in specific regions of the growth cone are involved in axon growth and guidance. For example, forward migration of the growth cone requires raft membranes in its leading front. Recent experiments have suggested that lipid rafts function as a platform for localized signaling downstream of adhesion molecules and guidance receptors. The rafts assemble into an active membrane domain that captures and reorganizes the cytoskeletal machinery. In this way, the spatial control of signaling through raft membranes plays a critical role in translating extracellular information into polarized motility of the growth cone.     

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

Spectrins: A structural platform for stabilization and activation of membrane channels,receptors and transporters

This review focuses on structure and functions of spectrin as a major component of the membrane skeleton. Recent advances on spectrin function as an interface for signal transduction mediation and a number of data concerning interaction of spectrin with membrane channels, adhesion molecules, receptors and transporters draw a picture of multifaceted protein. Here, we attempted to show the current depiction of multitask role of spectrin in cell physiology. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling

Analyzing the dynamics of membrane proteins in the context of cellular signaling represents a challenging problem in contemporary cell biology. Lateral diffusion of lipids and proteins in the cell membrane is known to be influenced by the cytoskeleton. In this work, we explored the role of the actin cytoskeleton on the mobility of the serotonin_1A (5-HT_1A) receptor, stably expressed in CHO cells, and its implications in signaling. FRAP analysis of 5-HT_1AR-EYFP shows that destabilization of the actin cytoskeleton induced by either CD or elevation of cAMP levels mediated by forskolin results in an increase in the mobile fraction of the receptor. The increase in the mobile fraction is accompanied by a corresponding increase in the signaling efficiency of the receptor. Interestingly, with increasing concentrations of CD used, the increase in the mobile fraction exhibited a correlation of ∼0.95 with the efficiency in ligand-mediated signaling of the receptor. Radioligand binding and G-protein coupling of the receptor were found to be unaffected upon treatment with CD. Our results suggest that signaling by the serotonin_1A receptor is correlated with receptor mobility, implying thereby that the actin cytoskeleton could play a regulatory role in receptor signaling. These results may have potential significance in the context of signaling by GPCRs in general and in the understanding of GPCR-cytoskeleton interactions with respect to receptor signaling in particular.     

The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.     

Cellular magnesium homeostasis

Magnesium, the second most abundant cellular cation after potassium, is essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways, as attested by more than 1000 entries in the literature. Despite significant recent progress, however, our understanding of how cells regulate Mg²⁺ homeostasis and transport still remains incomplete. For example, the occurrence of major fluxes of Mg²⁺ in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli has been extensively documented. Yet, the mechanisms ultimately responsible for magnesium extrusion across the cell membrane have not been cloned. Even less is known about the regulation in cellular organelles. The present review is aimed at providing the reader with a comprehensive and up-to-date understanding of the mechanisms enacted by eukaryotic cells to regulate cellular Mg²⁺ homeostasis and how these mechanisms are altered under specific pathological conditions.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein

Ecdysteroid is a crucial steroid hormone in insects, especially during metamorphosis. Here, we show that the Drosophila membrane steroid binding protein (Dm_MSBP) is a novel structural homolog of the vertebrate membrane-bound receptor component for progesterone. Dm_MSBP exhibited binding affinity to ecdysone when expressed on the cell surface of Drosophila S2 cells. In S2 cells, the stable overexpression of Dm_MSBP suppressed the growth arrest triggered by 20-hydroxyecdysone and prevented the temporal activation of extracellular signal-regulated kinase proteins. These results suggest that Dm_MSBP is a membranous suppressor to ecdysteroid and blocks the signaling by binding it in extracellular fluid.     

Mitogen-activated protein kinase signaling is involved in suramin-induced neurite outgrowth in a neuronal cell line

Suramin is a well-known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers. Previous study showed that suramin is an activator of extracellular signal-regulated kinase (ERK1/2) signaling in several cell lines including Chinese hamster ovary cells, although the physiological relevance of this activation remains uncertain. Here, it was shown that suramin enhances neurite outgrowth concomitant with activation of ERK1/2 in Neuro-2a cells, a neuronal cell line. These neurite outgrowth and ERK1/2 activation were significantly inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase, as well as by activation of endogenous adenosine A2A receptors. The suramin-induced phosphorylation of ERK1/2 was also inhibited by inhibitors of Src family kinases. This attenuation of ERK1/2 activity was accompanied by a significant decrease in suramin-induced neurite outgrowth. These results suggest that suramin activates the Src/ERK1/2 signaling pathway that induces neurite outgrowth, both of which are negatively regulated by cAMP produced in response to activation of endogenous adenosine A2A receptors.     

Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins

Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.     

Entamoeba histolytica: inhibition of cellular functions by overexpression of EhGEF1, a novel Rho/Rac guanine nucleotide exchange factor

The molecular, biochemical, and cellular characterization of EhGEF1 protein is described. Complete cDNA sequence of 1890 bp revealed an open reading frame that encodes a protein of 69 kDa. EhGEF1 is constituted of Dbl homology domain, pleckstrin homology domain, and several putative regulation sites. Studies of guanine nucleotide exchange activity of EhGEF1 on several GTPases from Entamoeba histolytica and Homo sapiens showed preferential activation on EhRacG, suggesting that EhGEF1 protein could be involved in mechanisms related to actin cytoskeleton activation, cytokinesis, capping, and uroid formation in trophozoite. Confocal microscopy studies of pExEhNeo/HSV-tagged-EhGEF1-transfected cells showed that trophozoites stimulated with ConA, EhGEF1, and EhRacG were localized at plasma membrane. Cellular studies showed that F-actin content of pExEhNeo/HSV-tagged-EhGEF1-transfected trophozoites as well as cellular migration and cell damage capacity were significantly altered. The observations suggest that EhRacG was the principal target of EhGEF1 and that EhGEF1 may provide a link between F-actin dynamics and EhRacG signaling.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.