Inositol Lipid Binding and Membrane Localization of Isolated Pleckstrin Homology (PH) Domains

Abstract

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 C␦ 1 (PLC␦ 1 ) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLC␦ 1 PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP 3 ) 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 InsP 3 and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLC␦ 1 PH-GFP. The Nterminal ligand binding domain of the type I InsP 3 receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP 3 than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal ␣-helix and the short loop between the ␤6 -␤7 sheets of the PLC␦ 1 PH domain, in addition to its InsP 3 -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 PLC␦ 1 PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), 1 a minor phospholipid component of the plasma membrane, is a key regulator of several cellular processes. PI(4,5)P 2 is a precursor of important second messengers, such as the diffusible InsP 3 , which regulates Ca 2ϩ release from intracellular Ca 2ϩ stores, and the protein kinase C activator, diacylglycerol (1, 2). PI(4,5)P 2 is also phosphorylated by class I PI 3-kinases to form PI(3,4,5)P 3 , which controls membrane recruitment and the functions of several important signaling proteins (3). PI(4,5)P 2 itself is a regulator of a great variety of target molecules, including ion channels (4, 5) and several proteins that regulate actin polymerization and the cytoskeleton (see Ref. 6), providing a link between the plasma membrane and the cortical cytoskeleton (7). PI(4,5)P 2 has been implicated in several forms of membrane remodeling events, including the fusion of secretory vesicles with the plasma membrane (8), clathrin-mediated endocytosis (9 -11), and membrane recovery by endocytosis during neurotransmitter release (12) (also see Ref. 13 for a review). Such diverse functions must rely upon interaction of the lipid with a large number of regulatory molecules. Most proteins that bind PI(4,5)P 2 contain a sequence composed of basic residues that provide electrostatic interaction with the phosphate groups of the inositol ring (6). Recent advances revealing the three-dimensional structures of several protein motifs that bind phosphoinosi...

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP 3 R) ligand-binding domain releases Ca 2+ via endogenous IP 3 R channels

“…The N-terminal IP 3 binding region (224 -605) of the type I IP 3 R or the pleckstrin homology (PH) domain of the phospholipase C (PLC)-like p130 protein (18) was fused to the mRFP for expression in COS-7 cells. The latter was used as a control, because it also binds IP 3 , although with somewhat lower affinity, but bears no structural homology to the IP 3 R LBD (16). Fusion of these domains to f luorescent proteins allowed monitoring of both the expression levels and the localization of the proteins simultaneously with cytoplasmic Ca 2ϩ measurements with Fura-2.…”

“…The construction of rat p130PH and the LBD of human type 1 IP 3 R (224-605) fused to the C-terminal of GFP have been described (16). The same constructs were also created fused to monomeric red fluorescent protein (mRFP) by exchanging the GFP coding sequence with that of mRFP (17).…”

“…The ER lumen-targeted protein coded by the pEF͞Myc͞ER͞GFP vector (Invitrogen) was used to visualize the ER. The design, production, and purification of recombinant proteins, as well as the IP 3 binding assays performed on them, have been described (16 depleted by Chelex 100 (Bio-Rad) treatment] supplemented with 2 mM ATP, 10 mM phosphocreatine, and 20 units͞ml creatine phosphokinase. Single-cell fluorescence measurements were performed at room temperature in the above microscope system by using 360 nm as the excitation wavelength.…”

Phosphoinositides in Constitutive Membrane Traffic

“…At higher expression levels, they will interfere with the normal regulation of PIP production. With these limitations in mind, PtdIns(3)P has been detected primarily on endocytic membranes (32,92,318), PtdIns(4,5)P 2 at the plasma membrane (148,149,219,234,365) and on internal membranes enriched in lipid rafts (27,297), and PtdIns(4)P on the Golgi (13,201,372). PtdIns4K activity has been detected in cell fractions enriched in lysosomes (7,54,56).…”

The Oxysterol-binding Protein Homologue ORP1L Interacts with Rab7 and Alters Functional Properties of Late Endocytic Compartments

“…We therefore assayed the interaction of the ORP1L PHD with phosphoinositides using a vesicle pull-down assay in which phosphatidylcholine (PC) vesicles containing 1 mol% of different phosphoinositides were incubated with glutathioneSepharose containing either GST, GST-PHD(ORP1L), or GST-PHD(PLC␦) as a positive control. In this assay, the PLC␦ PHD with high-affinity for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (Varnai et al, 2002) pulled down ϳ20% of the PI(4,5)P 2 -containing vesicles and also bound phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] and phosphatidylinositol 3,4,5-trisphosphate weakly [PI(3,4,5)P 3 ]. The PHD of ORP1L interacted weakly with PI(3,4)P 2 , phosphatidylinositol 3,5-bisphosphate, and PI(3,4,5)P 3 , and binding was also detectable for phosphatidylinositol 3-monophosphate, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 5-monophosphate (Figure 8).…”