Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

Abstract

The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2’s association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

Identification of gene regulation patterns underlying both oestrogen- and tamoxifen-stimulated cell growth through global gene expression profiling in breast cancer cells

“…A plasma-membrane-associated protein, EHD2, is found to regulate trafficking from the plasma membrane by controlling Rho GTPases activity (37). EHD proteins are also able to bind to phospholipids in plasma membrane (38, 39) and form complexes with IGF-1R to regulate downstream Akt pathway (40). We have found that IGF-1R and c-Src are tightly linked to the PI3K/Akt pathway in MCF-7:PF cells (15).…”

Cavin family proteins and the assembly of caveolae

“…Given the fact that between 15 and 20 cavin trimers are thought to be present in each caveola vesicle (Gambin et al, 2014), and that each trimer will possess at least two independent phospholipid-binding sites, membrane association and polymerisation of cavins are likely to, in turn, enhance the localised enrichment of PtdSer and PtdIns(4,5)P 2 observed at caveolae. This suggests a cooperative feedback mechanism between association of cavins with negatively charged phospholipids and increased localised concentrations of those lipids, which may then be important in regulating other caveola-associated factors that are involved in signalling and transport (Izumi et al, 1997;Simone et al, 2013). Overall, however, despite the clear activity of cavins in phospholipid binding, the actual role of these lipids in caveolar function remains quite poorly studied.…”

“…Caveolae have long been suggested to be important in binding to various membrane lipids and regulating their organisation in microdomains, including cholesterol, PtdSer and PtdIns(4,5)P 2 Fairn et al, 2011;Fujita et al, 2009;Murata et al, 1995;Parton and del Pozo, 2013;Pike and Casey, 1996;Yang et al, 2014). The affinity of these lipids for caveolae is reciprocated in the sense that depletion of either cholesterol or PtdIns(4,5)P 2 can, in turn, lead to loss of caveolae or dissociation of caveolar proteins (Breen et al, 2012;Rothberg et al, 1992;Simone et al, 2013). Liposome-based experiments suggest that the caveolin 'scaffolding domain', a 20-residue sequence of basic and hydrophobic amino acids, possesses an intrinsic ability to locally increase cholesterol, PtdSer and PtdIns(4,5)P 2 to a concentration several fold higher than in the surrounding membrane (Wanaski et al, 2003).…”

“…We imagine this to be a type of 'electrostatic trapping' of the cavin complex on the membrane. The resulting elevated concentration of phospholipids may also be required for localisation of other protein components, such as EHD2 (Simone et al, 2013), or enzymatic activities (Izumi et al, 1997). Phospholipid redistribution caused by caveola formation was also reported to modulate lipid-raft-associated Ras signalling .…”

The function of membrane-associated molecules in acquired resistance to antiestrogens in breast cancer

“…EHD2 is found to regulate trafficking from the plasma membrane by controlling Rho GTPases activity [19] . EHD proteins also have the capacity to associate with phospholipids in plasma membrane [23,24] and form complexes with IGF-1R to regulate downstream Akt pathway [25] . The LIM domain protein, FHL2, interacts with filamin A to remodel the cytoskeleton and acts as a novel coactivator in regulating target genes [20] .…”