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J. Biol. Chem., Vol. 279, Issue 23, 24362-24371, June 4, 2004

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The Pleckstrin Homology Domain of Phosphoinositide-specific Phospholipase C₄ Is Not a Critical Determinant of the Membrane Localization of the Enzyme^*

Sang Bong Lee, Péter Várnai¶||, Andras Balla, Kees Jalink**, Sue-Goo Rhee, and Tamas Balla

From the Endocrinology and Reproduction Research Branch, NICHHD, Laboratory of Cell Signaling, NHLI, National Institutes of Health, Bethesda, Maryland 20892, the ^¶Department of Physiology, Semmelweis University, Faculty of Medicine, Budapest, Hungary, H-1444 and ^**Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase C₄ (PLC₄) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLC₁ 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 PLC₄ PH domain was membrane-bound than in the case of PLC₁PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but a lower binding of PLC₄PH to lipid vesicles containing PI(4,5)P₂ was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) were required to displace the PLC₄PH from the lipid vesicles, and a lower Ins(1,4,5)P₃ affinity of PLC₄PH was found in direct Ins(1,4,5)P₃ binding assays. In sharp contrast to the localization of its PH domain, the full-length PLC₄ 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 PLC₁. A truncated PLC₄ 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 PLC₄ enzyme still showed binding to PI(4,5)P₂-containing micelles, but Ins(1,4,5)P₃ was significantly less potent in displacing the enzyme from the lipid than with the PLC₁ protein. These data suggest that although structurally related, PLC₁ and PLC₄ are probably differentially regulated in distinct cellular compartments by PI(4,5)P₂ and that the PH domain of PLC₄ does not act as a localization signal.

Received for publication, November 21, 2003 , and in revised form, March 15, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported in part by the Hungarian National Science Foundation Grant OTKA, T-034606.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 49, Rm. 6A35, 49 Convent Dr., Bethesda, MD 20892-4510. Tel.: 301-496-2136; Fax: 301-480-8010; E-mail: tambal{at}box-t.nih.gov.

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