Jill B. Jensen

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

The CB1 cannabinoid receptor mediates many of the psychoactive effects of Δ9THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB1/CB2 receptors may contribute to the behavioral, vascular, and immunological actions of Δ9THC 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 (Δ9THC, 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 Gq, G12, RhoA, actin, phospholipase C, and calcium release from IP3R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB1 and CB2.

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 (PIP2) 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 PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 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 M1 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 PIP2 synthesis and KCNQ interaction.

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. M1 muscarinic receptor (M1R) activation couples through Gαq to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-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 M1R activation, M1R/Gβ interaction, Gαq/Gβ separation, Gαq/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gαq/Gβ separation and Gαq/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 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 Gαq/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

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 (PIP2) 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 PIP2 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 M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

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 α1A-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 Gα subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Gα. Combining our data with a predictive Class A GPCR/Gα model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Gα. Such an interaction could promote opening of switch II of Gα to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

SYMPOSIUM REVIEW: 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 (PIP2) 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 PIP2 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 M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

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

Dickson et al. find that the ER membrane lipid phosphatase Sac1 localizes to ER–plasma membrane (PM) contact sites and acts as a cellular sensor and controller of PM phosphoinositide homeostasis.

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

Significance Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a key informational phospholipid, localized to and defining the inner leaflet of the plasma membrane (PM). How PM PI(4,5)P2 is sourced and regulated is critically important to the understanding of cellular trafficking, cell motility, membrane identity, and ion channel activity. The immediate precursor of PI(4,5)P2 is PI(4)P. Direct evidence detailing the location and contribution of PI(4)P pool(s) maintaining steady-state PM PI(4,5)P2 is lacking. We find that PM PI(4,5)P2 levels are supported by at least two continuously supplying precursor pools of PI(4)P, one in the PM and the other in the Golgi. The contribution of the Golgi pool of PI(4)P highlights the possibility that PM PI(4,5)P2 production is coupled to important cell biological processes. 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.

Novel probes for G-protein-coupled receptor signaling.

G-protein-coupled receptors (GPCRs) constitute the largest known protein family. They are activated by a wide range of ligands to transduce an extracellular signal into an intracellular one that evokes a variety of physiological responses, including regulation of ion channels by neurotransmitters

Lipid Signaling and Ion Channels

Many ion channels and ion transporters of the plasma membrane require the phosphoinositide phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) for full activity. This phosphoinositide is most prevalent in the plasma membrane and rare in other membranes. PIP 2 can be depleted by stimulating cell-surface receptors that activate phospholipase C, providing one pathway by which receptors can decrease activity of PIP 2 -sensitive channels. In addition, the requirement for PIP 2 may provide a mechanism to restrict activity of these channels to the plasma membrane.