Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C δ1 and p130

P. Várnai, Xuena Lin, Sang Bong Lee, Galina Tuymetova, Tzvetanka Bondeva, A. Spät, Sue Goo Rhee, György Hajno Czky, Tamas Balla

Research output: Contribution to journalArticle

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Abstract

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cδ1 (PLCδ1) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCδ1PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP3) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP3 and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCδ1PH-GFP. The N-terminal ligand binding domain of the type I InsP3 receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP3 than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal a-helix and the short loop between the β6-β7 sheets of the PLCδ1PH domain, in addition to its InsP3-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCδ1PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

Original languageEnglish
Pages (from-to)27412-27422
Number of pages11
JournalJournal of Biological Chemistry
Volume277
Issue number30
DOIs
Publication statusPublished - Jul 26 2002

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Type C Phospholipases
Inositol
Membrane Lipids
Green Fluorescent Proteins
Membranes
Lipids
Inositol Phosphates
Phosphatidylinositols
Mutagenesis
Amino Acid Motifs
Inositol 1,4,5-Trisphosphate
Collodion
Insertional Mutagenesis
Cell membranes
Programmable logic controllers
Pleckstrin Homology Domains
platelet protein P47
Proteins
Fusion reactions
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C δ1 and p130. / Várnai, P.; Lin, Xuena; Lee, Sang Bong; Tuymetova, Galina; Bondeva, Tzvetanka; Spät, A.; Rhee, Sue Goo; Czky, György Hajno; Balla, Tamas.

In: Journal of Biological Chemistry, Vol. 277, No. 30, 26.07.2002, p. 27412-27422.

Research output: Contribution to journalArticle

Várnai, P. ; Lin, Xuena ; Lee, Sang Bong ; Tuymetova, Galina ; Bondeva, Tzvetanka ; Spät, A. ; Rhee, Sue Goo ; Czky, György Hajno ; Balla, Tamas. / Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C δ1 and p130. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 30. pp. 27412-27422.
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AU - Lin, Xuena

AU - Lee, Sang Bong

AU - Tuymetova, Galina

AU - Bondeva, Tzvetanka

AU - Spät, A.

AU - Rhee, Sue Goo

AU - Czky, György Hajno

AU - Balla, Tamas

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AB - The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cδ1 (PLCδ1) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCδ1PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP3) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP3 and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCδ1PH-GFP. The N-terminal ligand binding domain of the type I InsP3 receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP3 than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal a-helix and the short loop between the β6-β7 sheets of the PLCδ1PH domain, in addition to its InsP3-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCδ1PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

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