Robin F. Irvine

Back in the water: The return of the inositol phosphates

Following the discovery of inositol-1,4,5-trisphosphate as a second messenger, many other inositol phosphates were discovered in quick succession, with some understanding of their synthesis pathways and a few guesses at their possible functions. But then it all seemed to go comparatively quiet, with an explosion of interest in the inositol lipids. Now the water-soluble phase is once again becoming a focus of interest. Old and new data point to a new vista of inositol phosphates, with functions in many diverse aspects of cell biology, such as ion-channel physiology, membrane dynamics and nuclear signalling.

PI4P and PI(4,5)P2 Are Essential But Independent Lipid Determinants of Membrane Identity

Phosphoinositide Contributions To study the roles of phosphoinositides in the plasma membrane of mammalian cells, Hammond et al. (p. 727, published online 21 June; see the Perspective by Fairn and Grinstein) engineered phosphatase molecules that could be targeted to the membrane on demand, where they would alter the concentrations of the phospholipids phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and phosphatidylinositol 4-phosphate (PI4P). PI4P was thought to provide a major source for the synthesis of PI(4,5)P2, but depletion of PI4P did not have much affect on synthesis of PI(4,5)P2. Instead, PI4P appears to help to establish the negative charge at the membrane and thus promote electrostatic interactions with positively charged amino acids in membrane-associated proteins and influencing function of ion channels. The phospholipid phosphatidylinositol 4-phosphate defines important physical properties of the cell membrane. The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P2 synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P2. However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P2, which may be fulfilled by a more general polyanionic lipid requirement.

Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.

Reduction of epidermal growth factor receptor affinity by heterologous ligands: Evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.

Inositol (1,4,5)trisphosphate-promoted Ca2+ release from microsomal fractions of rat liver.

Crude mitochondrial fractions containing a substantial amount of microsomes accumulate Ca2+ in the presence of ATP, ruthenium red and oligomycin. A proportion of this accumulated Ca2+ is released by the addition of low concentrations (ca. 1 microM) of inositol (1,4,5) trisphosphate . Under some conditions the release is transient, and evidence is presented which suggests that this is due to inhomogeneity in the vesicle population. (1,4,5)inositol trisphosphate -induced Ca2+ release can also be demonstrated, under appropriate experimental conditions, in a more purified microsomal fraction essentially free of mitochondria.

Effects of bombesin and insulin on inositol (1,4,5) trisphosphate and inositol (1,3,4) trisphosphate formation in Swiss 3T3 cells

The effects of bombesin and insulin, separately and in combination, have been studied in Swiss mouse 3T3 cells. Bombesin caused a rapid transfer of 3H from the lipid inositol pool of prelabeled cells into inositol phosphates. Label in inositol tetrakisphosphate (InsP4) and in Ins1,4,5P3 and Ins1,3,4P3 rose within 10 sec of stimulation and that in Ins1,4P2, another InsP2 and InsP1, more slowly. Insulin, which had little effect on its own, increased the turnover of inositol lipids due to acute bombesin stimulation and also enhanced the DNA synthesis evoked by prolonged bombesin treatment. The results suggest that bombesin acting as a growth factor, uses inositol lipids as part of its transduction mechanism and that insulin acts synergistically to enhance both inositol phosphate formation and DNA synthesis.

PIPkins1, their substrates and their products: new functions for old enzymes.

The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.

Transfer of arachidonic acid between phospholipids in rat liver microsomes

Abstract Phosphatidylcholine and phosphatidylinositol labelled with radioactive oleic, arachidonic or linoleic acids in the 2-acyl position were prepared. Rat liver microsomes were incubated with either lysophosphatidylcholine or lyso-phosphatidylinositol and the opposite 2-acyl-labelled phospholipid, and were found to catalyse a transfer of fatty acids between the two phospholipids. This was shown to be a direct Co-enzyme A-mediated transfer that does not involve a free fatty acid intermediate (i.e. it is independent of phospholipase A 2 activity). Arachidonoyl transfer took place at about four times the rate of linoleoyl transfer; oleoyl transfer was not detectable. The role of direct arachidonoyl transfer to phosphatidylinositol in the controlled release of arachidonic acid for prostaglandin synthesis is discussed.

Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay

Phosphatidylinositol 5‐phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) [Rameh et al. (1997) Nature 390, 192–196]. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with [γ‐32P]ATP and recombinant PIPkin IIα, and quantifying the radiolabelled PtdInsP2 formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist‐stimulated synthetic mechanism.

Diacylglycerol potentiates phospholipase attack upon phospholipid bilayers: Possible connection with cell stimulation

Many phospholipases of the A or C types show a very limited ability to hydrolyse phospholipids such as phosphatidylcholine or phosphatidylinositol which adopt a bilayer form when hydrated. The addition of unsaturated 1,2 or 1,3 diacylglycerols to such substances produces a marked stimulation of the attack. In contrast, the hydrolysis of phosphatidylethanolamine, which normally exists in the hexagonal II structure, is not stimulated by diacylglycerol. It is suggested that the liberation of diacylglycerol which often occurs during cell stimulation, may play a part in activating the associated cascade of phospholipid reactions.