title: The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme.
abstract: The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.
authors.affiliation: Laboratory of Cell Signaling, NHLI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Sang Bong
authors.initials: SB
authors.lastname: Lee
authors.forename: Péter
authors.initials: P
authors.lastname: Várnai
authors.forename: Andras
authors.initials: A
authors.lastname: Balla
authors.forename: Kees
authors.initials: K
authors.lastname: Jalink
authors.forename: Sue-Goo
authors.initials: SG
authors.lastname: Rhee
authors.forename: Tamas
authors.initials: T
authors.lastname: Balla
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D054803:Phospholipase C delta; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry*
chemical_list: D001798:Blood Proteins; D007527:Isoenzymes; D008055:Lipids; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D010738:Type C Phospholipases; D054803:Phospholipase C delta