Varnai, P. et al. Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130. J. Biol. Chem. 277, 27412-27422

Semmelweis University, Budapeŝto, Budapest, Hungary
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2002; 277(30):27412-22. DOI: 10.1074/jbc.M109672200
Source: PubMed


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 α-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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Available from: Gyorgy Hajnoczky
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