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Anjaparavanda P Naren

Publications and source records attributed to Anjaparavanda P Naren.

16 recordsLinked to original sources
PubMed2006

Assembly of an SAP97-AKAP79-cAMP-dependent protein kinase scaffold at the type 1 PSD-95/DLG/ZO1 motif of the human beta(1)-adrenergic receptor generates a receptosome involved in receptor recycling and networking.

Appropriate trafficking of the beta(1)-adrenergic receptor (beta(1)-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the beta(1)-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the beta(1)-AR (Gardner, L. A., Tavalin, S. A., Goehring, A., Scott, J. D., and Bahouth, S. W. (2006) J. Biol. Chem. 281, 33537-33553). In this study we show that AKAP79 forms a complex with the type 1 PDZ-binding sequence (ESKV) at the extreme carboxyl terminus of the beta(1)-AR, which is mediated by the membrane-associated guanylate kinase (MAGUK) protein SAP97. Thus, the PDZ and its associated SAP97-AKAP79 complex are involved in targeting the cyclic AMP-dependent protein kinase (PKA) to the beta(1)-AR. The PDZ and its scaffold were required for efficient recycling of the beta(1)-AR and for PKA-mediated phosphorylation of the beta(1)-AR at Ser(312). Overexpression of the catalytic subunit of PKA or mutagenesis of Ser(312) to the phosphoserine mimic aspartic acid both rescued the recycling of the trafficking-defective beta(1)-ARDelta PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the beta(1)-AR to Ser(312) in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the beta(1)-AR. The data presented here show that a novel beta(1)-adrenergic receptosome is organized at the beta(1)-AR PDZ to generate a scaffold essential for trafficking and networking of the beta(1)-AR.

A Kinase Anchor Proteins↗
PubMed2006

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Animals↗
PubMed2006

MAPK interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide.

The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pair-wise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF.

Caco-2 Cells↗
PubMed2005

Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

Analysis of Variance↗
PubMed2005

Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners.

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.

Animals↗
PubMed2004

Protease-activated receptor-1 activation of endothelial cells induces protein kinase Calpha-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression.

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Calpha activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Calpha activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.

Animals↗
PubMed2004

Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation.

Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (DeltaF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented DeltaF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not DeltaF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR.

Animals↗
PubMed2004

Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.

Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis trans-membrane conductance regulator (CFTR) Cl(-) channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute approximately 2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signaling-related proteins such as Gbeta and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.

Amino Acid Motifs↗
PubMed2004

ENaC subunit-subunit interactions and inhibition by syntaxin 1A.

Amiloride-sensitive epithelial Na(+) channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity. Syntaxin 1A was able to coimmunoprecipitate in vitro-translated gamma-ENaC, but not alpha- or beta-ENaC. Also, using antibodies raised against alpha-, beta-, or gamma-ENaC, we detected syntaxin 1A in immunoprecipitates from Madin-Darby canine kidney cells stably transfected with alphabetagamma-ENaC. In bilayers, syntaxin 1A inhibited ENaC, and this syntaxin 1A modulation of ENaC activity was eliminated by truncations of cytoplasmic domains of the ENaC subunits. Our findings provide evidence for a direct physical interaction between ENaC and syntaxin 1A and suggest involvement of ENaC's cytoplasmic domains in functional modulation of ENaC activity by syntaxin 1A.

Animals↗
PubMed2003

Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions.

A recent study (Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C., and Mrsny, R. J. (2000) J. Biol. Chem. 275, 29816-29822) suggested that phosphatidylinositol 3-kinase (PI 3-kinase) may interact with occludin; however, there exists no evidence of direct interaction of PI 3-kinase with the tight junctions. Activation of PI 3-kinase by oxidative stress and its role in disruption of tight junctions was examined in Caco-2 cell monolayer. The oxidative stress-induced decrease in electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 were reduced by a PI 3-kinase inhibitor, LY294002. Oxidative stress-induced tyrosine phosphorylation and dissociation from the actin cytoskeleton of occludin and ZO-1 were reduced by LY294002. The regulatory subunit of PI 3-kinase, p85, and the PI 3-kinase activity were co-immunoprecipitated with occludin, which were rapidly increased by oxidative stress. Oxidative stress resulted in increased translocation of p85 from the intracellular compartment into the intercellular junctions. Pair-wise glutathione S-transferase pull-down assay showed that glutathione S-transferase-occludin (C-terminal tail) binds to recombinant p85. This study shows that oxidative stress increases the association of PI 3-kinase with the occludin, and that PI 3-kinase activity is involved in oxidative stress-induced disruption of tight junction.

Chromones↗
PubMed2003

Acid-sensing ion channels in malignant gliomas.

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.

Acid Sensing Ion Channels↗
PubMed2002

A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

Animals↗
PubMed2002

CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.

Animals↗
PubMed2002

Mechanisms of CFTR regulation by syntaxin 1A and PKA.

Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl(-) currents (I(CFTR)) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues: (a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (P(o)) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or syntaxin 1A association.

Antigens, Surface↗
PubMed2002

Quantal release of free radicals during exocytosis of phagosomes.

Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space.

Animals↗