The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

Jan, Chung Ren; Ho, Chin Man; Wu, Sheng‐Nan; Tseng, Ching Jiunn

doi:10.1016/s0024-3205(98)00346-4

Cited by 53 publications

(42 citation statements)

References 26 publications

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“…correlates with the described capacity of U73122 to induce the release of Ca 2ϩ from intracellular deposits (27). Altogether, these data suggest that the C2A domain of JFC1 may be dissociated from the membrane by calcium-mediated electrostatic interference rather that by a Ca 2ϩ -mediated PLC activation.…”

Section: Does the Jfc1 C2a Domain Bind To Ptdins(45)p2 In Living Cells?supporting

confidence: 72%

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Catz

Johnson

Babior

2002

Proc. Natl. Acad. Sci. U.S.A.

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Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3 -phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3 -phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3 -phosphoinositides in vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3 -phosphoinositides in vitro with similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Akt in vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking. P hosphoinositides are specialized phospholipids known to localize to the inner leaflet of the plasma membrane, vesicles, and the nuclear envelope and participate in several signaling pathways (1). One of these phosphoinositides, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ], plays a central role in the regulation of several intracellular pathways, from actin cytoskeleton modulation and vesicle trafficking (2) to modulation of the transcription factor Tubby protein (3). Furthermore, PtdIns(4,5)P 2 is a precursor for the second messenger phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P 3 ] via the phosphoinositide 3-kinases ref. 4). PI 3-kinases and their products 3Ј-phosphorylated phosphoinositides (3Ј-phosphoinositides) are also involved in many crucial cell functions including vesicle trafficking (1, 2), cytoskeleton modulation, and cell survival (5). It is now clear that most of the functions regulated by phosphoinositides are mediated by adaptor proteins that share the ability to bind to PtdIns(4,5)P 2 and PI 3-kinase products with high affinity and specificity. These effectors are characterized by the presence of specialized phosphoinositide-binding domains. These domains include the pleckstrin homology (PH) domain (6), the FYVE finger (7), and the recently discovered PHOX homology (PX) domain (8). Furthermore, C2 domains have been shown to bind to phospholipids or phosphoinositides in the presence or absence of calcium (9). Proteins that contain C2 domains could generally be categorized as signal transduction mediators or membrane traffic effectors (10). The most extensively studied proteins included in the last group are the synaptotagmins (Syts). Syts are a group of transmembrane proteins that contain tan...

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Section: Does the Jfc1 C2a Domain Bind To Ptdins(45)p2 In Living Cells?supporting

confidence: 72%

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Catz

Johnson

Babior

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…4). This concentration of U73122 has previously been shown to be specific for PLC (55) and does not increase intracellular Ca 2ϩ , whereas higher concentrations of U73122 inhibit PLC and also lead to a release of Ca 2ϩ from intracellular stores (30). The PLC inhibitor U73122 suppressed Env-dependent cell-cell fusion by an average of 73% Ϯ 7.6% (Fig.…”

Section: Resultsmentioning

confidence: 75%

“…PBLs were pretreated with U73122, U73343, and TAK-779 for 1 h prior to mixture with BSC40 cells expressing different HIV-1 Env subtypes and measurement of cell-cell fusion (30). The PLC inhibitor U73122 suppressed Env-dependent cell-cell fusion in PBLs by an average of 74% Ϯ 12.8%, and U73343 had ATPase (SERCA) pumps, were used to deplete internal Ca 2ϩ stores.…”

Section: Resultsmentioning

confidence: 99%

Induction of the Gα_qSignaling Cascade by the Human Immunodeficiency Virus Envelope Is Required for Virus Entry

Harmon

Ratner

2008

J Virol

109

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Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of G␣ q and its downstream targets. Fusion and Rac-1 activation are mediated by G␣ q and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting G␣ q or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca 2؉ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for G␣ i or G␣ s signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the G␣ q pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance.Entry of human immunodeficiency virus type 1 (HIV-1) is mediated by sequential binding of the trimeric HIV envelope glycoprotein (Env) to CD4 and one of two primary chemokine coreceptors, CXCR4 or CCR5. This binding triggers a series of conformational changes in Env that expose the fusion peptide, which then induces the merger of viral or infected cell membranes with target membranes (14,24,53). The HIV-1 Env interaction with CD4 and a coreceptor also stimulates various intracellular signaling events similar to those initiated by their natural ligands, such as phosphorylation of Pyk2, Ca 2ϩ mobilization, activation of RhoGTPases, and actin cytoskeleton rearrangements (15,20,36,37,48,51,53). Actin cytoskeletal remodeling and RhoGTPases play a central role in regulating fusion of biological membranes (13,22). In the case of HIV-1-induced membrane fusion, activation of the RhoGTPase Rac-1 and subsequent actin cytoskeletal reorganizations are required for efficient virus entry and infection (29, 48). The exact mechanism of Env-induced Rac-1 activation that mediates actin cytoskeletal rearrangements and induces membrane fusion has not been investigated.Binding of both CD4 and the coreceptors elicits signaling pathways that result in Rac-1 activation. However, previous results suggest that Env-induced Rac-1 activation is mediated via the coreceptor rat...

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“…U73343, the inactive analogue of U73122, had no significant effect upon the release of Ca 2 þ (P40.05, n ¼ 18). A dose of 1 mM U73122 was not exceeded as this compound has been shown to release Ca 2 þ from internal stores at higher doses (Willems et al, 1994;Mogami et al, 1997;Jan et al, 1998 ( Figure 4a). These results suggest that the observed WIN55,212-2-mediated Ca 2 þ mobilization did not depend on CB 1 -G i/o coupling.…”

Section: Cb 1 Expression In Htm Cellsmentioning

confidence: 99%

Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

McIntosh

Hudson

Yegorova

et al. 2007

British J Pharmacology

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Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB 1 receptors (CB 1 ) are present in TM and cannabinoid administration decreases IOP. CB 1 signalling was investigated in a cell line derived from human TM (hTM). Experimental approach: CB 1 signalling was investigated using ratiometric Ca 2 þ imaging, western blotting and infrared In-Cell Western analysis. Key results: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 mM) increased intracellular Ca 2 þ in hTM cells. WIN55,212-2-mediated Ca 2 þ increases were blocked by AM251, a CB 1 antagonist, but were unaffected by the CB 2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca 2 þ ] i was pertussis toxin (PTX)-insensitive, therefore, independent of G i/o coupling, but was attenuated by a dominant negative Ga q/11 subunit, implicating a G q/11 signalling pathway. The increase in [Ca 2 þ ] i was dependent upon PLC activation and mobilization of intracellular Ca 2 þ stores. A PTXsensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a G i/o signalling pathway. CB 1 -G q/11 coupling to activate PLC-dependent increases in Ca 2 þ appeared to be specific to WIN55,212-2 and were not observed with other CB 1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca 2 þ ] i at concentrations Z15 mM, and PTX-sensitive increases in ERK1/2 phosphorylation. Conclusions and implications: This study demonstrates that endogenous CB 1 couples to both G q/11 and G i/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB 1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.

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The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

Cited by 53 publications

References 26 publications

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Induction of the Gα_qSignaling Cascade by the Human Immunodeficiency Virus Envelope Is Required for Virus Entry

Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

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The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

Cited by 53 publications

References 26 publications

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Induction of the GαqSignaling Cascade by the Human Immunodeficiency Virus Envelope Is Required for Virus Entry

Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

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Induction of the Gα_qSignaling Cascade by the Human Immunodeficiency Virus Envelope Is Required for Virus Entry