Localization and Turnover of Phosphatidylinositol 4,5-Bisphosphate in Caveolin-enriched Membrane Domains

Abstract

Caveolae are small, plasma membrane invaginations that have been implicated in cell signaling. In A431 cells, approximately half of the total cellular phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) was found to be localized in low density, Triton-insoluble membrane domains enriched in caveolin. Treatment of cells with either epidermal growth factor or bradykinin for 5 min at 37°C resulted in approximately a 50% decrease in this caveolar PtdIns 4,5-P 2 with no change in the levels of plasma membrane PtdIns 4,5-P 2 . These data suggest that the PtdIns 4,5-P 2 present in cells is largely compartmentalized and that the caveolar PtdIns 4,5-P 2 is subject to hydrolysis by hormone-stimulated phospholipase C. As growth factor receptors, seven transmembrane domain receptors, heterotrimeric G proteins, and the inositol trisphosphate receptor have all been shown to be enriched in caveolae, these findings suggest that both the generation and response to inositol trisphosphate is highly compartmentalized within the cell.Caveolae are small, plasma membrane invaginations that are involved in apical protein sorting (1, 2) and the uptake of folates by potocytosis (3). In most cell types, they account for 1% or less of the total plasma membrane (4). Caveolin, a 21-kDa substrate for pp60 src (5), is localized almost exclusively to caveolae and probably represents a structural component of this plasma membrane domain (6 -8). Caveolae are also enriched in glycosphingolipids and cholesterol, making these domains resistant to extraction in Triton X-100 (1).Caveolae have been shown to contain a variety of molecules involved in cell signaling including low molecular weight and heterotrimeric G proteins (9, 10), Src family kinases (10), mitogen-activated protein kinase (10), the epidermal growth factor receptor (11,12), and the platelet-derived growth factor receptor (13). These findings implicate caveolae in signal transduction and suggest that many of the molecular components for cell signaling are localized in this relatively small area of the plasma membrane.We have recently shown that in Madin Darby canine kidney cells, a large proportion of the total cellular PtdIns 4,5-P 2 1 resides in detergent-insoluble lipid domains enriched in caveolin (14). Given this observation, the question arises as to whether the PtdIns 4,5-P 2 present in caveolae is subject to turnover in response to growth factors and hormones. We now report that, in A431 cells, at least 50% of the PtdIns 4,5-P 2 is present in caveolae, and this pool of phospholipid is reduced in cells treated with EGF or bradykinin. Polyphosphoinositides present in the plasma membrane fraction are not altered by hormone treatment. These findings suggest that PtdIns 4,5-P 2 is highly compartmentalized within cells and that caveolae are the primary site of agonist-stimulated PtdIns 4,5-P 2 turnover. EXPERIMENTAL PROCEDURESMaterials-Anti-caveolin antibodies were from Transduction Laboratories (Lexington, KY). Anti-actin antibodies were from Chemicon (El Segundo, CA). The polyclo...

Identification and Characterization of a Novel Plant Phospholipase D That Requires Polyphosphoinositides and Submicromolar Calcium for Activity in Arabidopsis

“…However, PIP 2 is likely to be more concentrated in the inner leaflet of plasma membranes. In addition, there is a possibility for the occurrence of PIP 2 -enriched membrane microdomains, as those domains have been reported in mammalian cells (30). Furthermore, this new PLD is also stimulated by PIP.…”

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

“…Notably, the identified interaction in this report is reminiscent of the interaction between the caveolar Trp channels and the ER-localized IP3 receptors (Kiselyov et al, 1999;Lockwich et al, 2001). In addition, several studies have shown that caveolae not only contain PLC-␥1, but also 50% of the plasma membrane PIP2, a substrate of PLC-␥1 (Pike and Casey, 1996;Jang et al, 2001).…”

The mechanism of phospholipase C-γ1 regulation

“…Moreover, the larger proportion of PIP 2 in cells is localized in caveolin-enriched micro-domains (caveoli) (Pike and Casey, 1996). Caveoli are known to be signal initiation sites where many intracellular signaling molecules, such as EGF receptor, G-α subunit, ras, etc.…”

“…However, until now, a protein tyrosine phosphatase acting on tyrosyl phosphorylated PLC-γ1 has not been identified. Discovery of an in vivo protein tyrosine phosphatase for PLC-γ1 would widely extend our knowledge of the regulatory mechanism of PLC-γ1.Recently we have identified that PTP-1B, a cytosolic protein tyrosine phosphatase, can dephosphorylate PLC-γ1 in vitro and vvivo (Kim et al, unpublished data).Moreover, the larger proportion of PIP 2 in cells is localized in caveolin-enriched micro-domains (caveoli) (Pike and Casey, 1996). Caveoli are known to be signal initiation sites where many intracellular signaling molecules, such as EGF receptor, G-α subunit, ras, etc.…”