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Cited by 859Protein Kinase Regulation and GTPase SignalingCellular transport and secretionLipid metabolism and biosynthesis

Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

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Várnai, Péter, Balla, Tamás·1998·The Journal of Cell Biology
DOI 10.1083/jcb.143.2.501PMC PMC2132833
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Abstract

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

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  • PLCδ-PH-GFP can be used not only to monitor the distribution of 4,5-PIP2, but when expressed at sufficiently high levels, also to competitively inhibit other 4,5-PIP2-dependent processes.

    “PLCδ-PH-GFP can be used not only to monitor the distribution of 4,5-PIP2, but when expressed at sufficiently high levels, also to competitively inhibit other 4,5-PIP2–dependent processes.”

    From Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis (2000) · cites this paper as Varnai and Balla 1998

    The Results section (‘PH Domains Can Inhibit the Agonist-induced Hydrolysis…’; page unavailable) reports that overexpressed PLCδ PH-domain–GFP greatly inhibited agonist-stimulated inositol phosphate formation. The Discussion proposes masking PtdIns(4,5)P2 or competing with PLC enzymes as possible mechanisms, but does not establish competitive inhibition or demonstrate inhibition of other PtdIns(4,5)P2-dependent processes beyond the PLC response tested. (LLM, conf=0.84)

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  • PLCδ-PH-GFP associated predominantly with the plasmalemma of RAW cells, with little binding to organellar membranes.

    “As reported for other cell types (Stauffer et al. 1998; Varnai and Balla 1998), PLCδ-PH-GFP associated predominantly with the plasmalemma of RAW cells, with little binding to organellar membranes (Fig. 1 A).”

    From Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis (2000) · cites this paper as Varnai and Balla 1998

  • PLCδ-PH-GFP and C1δ-GFP probes had been validated in other systems and their suitability was confirmed in RAW macrophages.

    “These probes had been validated in other systems (Oancea et al. 1998; Stauffer et al. 1998; Varnai and Balla 1998) and their suitability was confirmed in RAW macrophages.”

    From Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis (2000) · cites this paper as Varnai and Balla 1998

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Authors
Várnai, P., & Balla, T.
Affiliations
National Institutes of Health (US)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
1998-10-19
Volume
143
Issue
2
Pages
501-510
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.143.2.501
PMC
PMC2132833
OpenAlex
W2153841101
Cited by
859
References
1
Citations by year
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FWCI
6.76
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
Keywords
phosphatidylinositol 4,5-bisphosphate, pleckstrin homology domain, phosphoinositide binding, phospholipase C delta, GFP fusion protein, plasma membrane localization, calcium signaling, agonist stimulation, ionomycin, quercetin, phenylarsine oxide, wortmannin, inositol phospholipids
MeSH terms
Animals, Blood Proteins, Calcium, Cell Membrane, Chelating Agents, Egtazic Acid, Indicators and Reagents, Ionophores, Luminescent Proteins, Type C Phospholipases, Phosphoproteins, Protein Binding, Transfection, Tritium, Inositol 1,4,5-Trisphosphate, Ionomycin, 3T3 Cells, Protein Structure, Tertiary, Phosphatidylinositol 4,5-Diphosphate, COS Cells, Green Fluorescent Proteins, Mice

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