Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Dishevelled coordinates phosphoinositide kinases PI4KIIIα and PIP5KIγ for efficient PtdInsP2 synthesis.

Phosphatidylinositol(4,5)-bisphosphate (PtdInsP2) is an important modulator of many cellular processes, and its abundance in the plasma membrane is closely regulated. We examined the hypothesis that members of the Dishevelled scaffolding protein family can bind the lipid kinases phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), facilitating synthesis of PtdInsP2 directly from phosphatidylinositol. We used several assays for PtdInsP2 to examine the cooperative function of phosphoinositide kinases and the Dishevelled protein Dvl3 in the context of two receptor signaling cascades. Simultaneous overexpression of PI4KIIIα (also known as PI4KA) and PIP5KIγ (also known as PIP5K1C) had a synergistic effect on PtdInsP2 synthesis that was recapitulated by overexpression of Dvl3. Increasing the activity of Dvl3 by overexpression increased resting plasma membrane PtdInsP2. Knockdown of Dvl3 reduced resting plasma membrane PtdInsP2 and slowed PtdInsP2 resynthesis following receptor activation. We confirm that Dvl3 promotes coupling of PI4KIIIα and PIP5KIγ and show that this interaction is essential for efficient resynthesis of PtdInsP2 following receptor activation.

Plasma membrane processes are differentially regulated by type I phosphatidylinositol phosphate 5-kinases and RASSF4.

Phosphoinositide lipids regulate many cellular processes and are synthesized by lipid kinases. Type I phosphatidylinositol phosphate 5-kinases (PIP5KIs) generate phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Several phosphoinositide-sensitive readouts revealed the nonequivalence of overexpressing PIP5KIß, PIP5KIγ or Ras association domain family 4 (RASSF4), believed to activate PIP5KIs. Mass spectrometry showed that each of these three proteins increased total cellular phosphatidylinositol bisphosphates (PtdInsP2) and trisphosphates (PtdInsP3) at the expense of phosphatidylinositol phosphate (PtdInsP) without changing lipid acyl chains. Analysis of KCNQ2/3 channels and PH domains confirmed an increase in plasma membrane PtdIns(4,5)P2 in response to PIP5KIß or PIP5KIγ overexpression, but RASSF4 required coexpression with PIP5KIγ to increase plasma membrane PtdIns(4,5)P2 Effects on the several steps of store-operated calcium entry (SOCE) were not explained by plasma membrane phosphoinositide increases alone. PIP5KIß and RASSF4 increased STIM1 proximity to the plasma membrane, accelerated STIM1 mobilization and speeded onset of SOCE; however, PIP5KIγ reduced STIM1 recruitment but did not change induced Ca2+ entry. These differences imply actions through different segregated pools of phosphoinositides and specific protein-protein interactions and targeting.This article has an associated First Person interview with the first author of the paper.

Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play an integral role in cellular processes such as excitation-contraction coupling and store-operated calcium entry (SOCE). Another ER-PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER-PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2 Activation of G protein-coupled receptors that deplete PM PI(4,5)P2disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1's access to the PM and permitting PM PI(4)P and PI(4,5)P2to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER-PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.

Regulation of calcium and phosphoinositides at endoplasmic reticulum-membrane junctions.

Effective cellular function requires both compartmentalization of tasks in space and time, and coordination of those efforts. The endoplasmic reticulum's (ER) expansive and ramifying structure makes it ideally suited to serve as a regulatory platform for organelle-organelle communication through membrane contacts. These contact sites consist of two membranes juxtaposed at a distance less than 30 nm that mediate the exchange of lipids and ions without the need for membrane fission or fusion, a process distinct from classical vesicular transport. Membrane contact sites are positioned by organelle-specific membrane-membrane tethering proteins and contain a growing number of additional proteins that organize information transfer to shape membrane identity. Here we briefly review the role of ER-containing membrane junctions in two important cellular functions: calcium signalling and phosphoinositide processing.

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current.

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P2 pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PHPLCδ1 domains as real-time indicators of PM PI(4,5)P2, and translocation of PHOSH2×2, and PHOSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P2 measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P2. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P2. The decline of PI(4,5)P2 following 4-phosphatase recruitment takes 1-2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P2 appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P2 when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P2 production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.

Phosphoinositides: lipid regulators of membrane proteins.

Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP(2)) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP(2) to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K(+) channel current by activation of M(1) muscarinic receptors; KCNQ channels require PIP(2) for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells.

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP(2)) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP(2) pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP(2) with a time constant of tau <300 ms and fully inhibits KCNQ currents in a similar time. PIP(2) was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, tau = 11 s. In contrast, resynthesis of PIP(2) after activation of phospholipase C by muscarinic receptors took approximately 130 s. These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M(1) muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP(2) synthesis and KCNQ interaction.

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells.

G protein-coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Galpha subunit, are common for many different GPCRs. For G(q)-coupled M(1) muscarinic (acetylcholine) receptors (M(1)Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M(1)R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M(1)R signaling through G(q) to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP(2)-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with approximately 3,000 fluorescently labeled receptors, G proteins, or PLC molecules per microm(2) of plasma membrane. Endogenous levels are much lower, 1-40 per microm(2). Our kinetic model reproduces the time courses and concentration-response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M(1)R and Gbeta FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Galpha(q) greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Galpha(q) subunits bound to PLC.

Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels.

G protein-coupled receptors initiate signaling cascades. M(1) muscarinic receptor (M(1)R) activation couples through Galpha(q) to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of PIP(2) closes PIP(2)-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M(1)R activation, M(1)R/Gbeta interaction, Galpha(q)/Gbeta separation, Galpha(q)/PLC interaction, and PIP(2) hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M(1)R activation (<100 ms) and M(1)R/Gbeta interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Galpha(q)/Gbeta separation and Galpha(q)/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP(2) hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Galpha(q)/PLC interaction. Evidently, channel release of PIP(2) and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current.

The CB(1) cannabinoid receptor mediates many of the psychoactive effects of Delta(9)THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB(1)/CB(2) receptors may contribute to the behavioral, vascular, and immunological actions of Delta(9)THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Delta(9)THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve G(q), G(12), RhoA, actin, phospholipase C, and calcium release from IP(3)R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB(1) and CB(2).