Polyphosphoinositide binding domains: Key to inositol lipid biology

Abstract

Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation of many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions. In this review, we discuss current knowledge of the principle types of PPIn-protein interactions, focusing on specific lipid-binding domains. We then discuss how these domains have been re-tasked by biologists as molecular probes for these lipids in living cells. Finally, we describe how the knowledge gained with these probes, when combined with other techniques, has led to the current view of the lipids' localization and function in eukaryotes, focusing mainly on animal cells. This article is part of a Special Issue entitled Phosphoinositides.

Keywords: Fluorescence imaging; Membrane; PH-domain; PX-domain; Phosphoinositide; Phospholipase C.

Figure 1. PPIn and their cellular distribution

The structure of phosphatidylinositol (PtdIns) is shown with the hydroxyls 3–5 available for reversible phosphorylation indicated. The cartoon shows a generic mammalian cell with the major PPIn conversion reactions indicated in their native membranes. Note that PtdIns synthesis occurs in the endoplasmic reticulum (ER), from whence it is known to be transferred to the plasma membrane or Golgi as depicted.

Figure 2. Five functional principles underlying PPIn-regulated protein function

(i) high affinity binding between protein and PPIn drives membrane binding (ii) co-incident binding between a protein, PPIn and a tertiary determinant (in this case a small G-protein, but it may also be a physical determinant such as membrane curvature) provide the avidity necessary for membrane binding. (iii) Polyvalent clustering of PPIn through non-specific electrostatic interactions with the polybasic region of a amphilic peptide motif contributes to membrane tragetting at anionic lipid-rich membrane (iv) binding of PPIn induces a conformational change in a binding protein. (v) PPIn interaction with a transfer protein facilitates lipid transfer.

Figure 3. specificity of two PtdIns(4,5)P₂ biosensors in cells

Depletion of PtdIns(4,5)P₂ at the PM causes release of Plcd4-PH-GFP, showing the dependence on this lipid for probe localization. Alternatively, driving ectopic synthesis of PtdIns(4,5)P₂ at Rab7-labelled membranes is sufficient to recruit the PtdIns(4,5)P₂ biosensors PH-Plcd4-GFP and Tubby_c-GFP. The indicated biosensors fused to GFP were expressed in COS-7 cells with either an FRB-PM motif (N-terminal peptide from Lyn kinase) and FKBP fused to the INPP5E 5-phosphatase, or an FRB-fused Rab7 and an FKBP-fused PIP5K. Addition of rapamycin induced dimerization of FRB and FKBP, leading to recruitment of the enzyme to the labelled compartment and depletion or synthesis of PtdIns(4,5)P₂, respectively. Insets show enlarged view of the boxed region.

Figure 4. quantitative aspects of a PtdIns(4,5)P₂ biosensor

A. The fraction of PH-PLCD1-GFP expected to be membrane bound in a cell with varying PtdIns(4,5)P₂ concentrations. For SH-SY5Y cells the fraction bound (~64%, red line) was estimated after treating cells with ionomycin to cause complete release of the probe into the cytosol, giving the estimated [PtdIns(4,5)P₂] = 3.5 μM. B. Three SH-SY5Y cells expressing PH-PLCD1-GFP, either at rest of after activation of endogenous cholinergic M3 receptors with carbachol. C. Carbachol treatment is known to induce ~85% depletion of PtdIns(4,5)P₂, and the theoretical and experimentally-measured change in cytosolic fraction of biosensor is shown. See text for details.