The CB1 cannabinoid receptor mediates many of the psychoactive effects of ⌬ 9 THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB 1/CB2 receptors may contribute to the behavioral, vascular, and immunological actions of ⌬ 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 (⌬ 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, G12, RhoA, actin, phospholipase C, and calcium release from IP 3R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB 1 and CB2.orphan ͉ pain ͉ CB3 ͉ G protein-coupled receptor C annabis has been used and abused for its therapeutic and psychoactive properties for millennia. The effects of cannabinoid compounds are largely mediated by cannabinoid receptors. CB 1 , cloned in 1990 (1), is widely and highly expressed in the CNS, where it likely mediates the majority of the psychotropic and behavioral effects of cannabinoids. CB 2 is primarily expressed in peripheral tissues (2). Both CB 1 and CB 2 are 7-transmembrane G protein-coupled receptors that engage predominantly the G i/o family of G proteins. However, ample evidence suggests that additional receptors may contribute to the behavioral, vascular, and immunological actions of ⌬ 9 tetrahydrocannabinol (THC) and endogenous cannabinoids (3).It has been suggested that GPR55 is a novel cannabinoid receptor (reviewed in ref. 4). GPR55 is only 13.5% identical to CB 1 and 14.4% identical to CB 2 , and its mRNA is present in the brain and periphery (5-7). A recent study found that a variety of cannabinoid compounds stimulated GTP␥S binding in cells stably expressing GPR55 (6). Here, we report GPR55 activation by THC, JWH015, and anandamide increases intracellular calcium by activating signaling pathways quite distinct from those used by CB 1 and CB 2 . Results Activation of GPR55 by Cannabinoids Increases Intracellular Calcium.We first examined the signaling pathways activated by GPR55 in HEK293 cells transiently expressing human GPR55 (hGPR55). Perfusion with 5 M THC evoked a calcium increase (⌬[Ca 2ϩ ] i ) averaging Ϸ100 nM (n ϭ 7, Fig. 1 A and B). Perfusion with 3 M THC evoked a more modest increase (n ϭ 5, 50 nM; Fig. 1B). The agonist-induced calcium response was present in all cells tested, but because it varied in magnitude and time course, concurrent controls were always conducted. GPR55 was essential for the THC-evoked calcium rise because there was minimal calcium rise in nontransfected HEK293 cells exposed to 5 M THC (n ϭ 6, Fig. 1 A and B). A similar calcium increase was seen in CHO cells stably expressing hGPR55 (data not show...

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.

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 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. Phosphoinositide structurePhosphoinositides are minority phospholipids of all eukaryotic cellular membranes. Like other phospholipids they have a glycerol backbone esterified to two fatty acid chains and a phosphate, and attached to a polar head group that extends into the cytoplasm (Fig. 1A). For phosphoinositides, the head group is the cyclic polyol myo-inositol, (CHOH) 6 . This inositol head group has free hydroxyl groups at positions D2 through D6, and those at positions D3, D4 and D5 are readily phosphorylated by cytoplasmic lipid kinases. This essay discusses the concept that the resulting seven combinatorially phosphorylated forms (Fig. 1B) of the inositol head group have informational content. Rather than playing a significant structural role in the lipid bilayer, polyphosphoinositides serve both as acidic address labels that identify different membranes and as instructions for This review was

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 Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), 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 M1R 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 M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-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 ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. 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 M1R and Gβ 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 Gαq 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 Gαq subunits bound to PLC.

Abbreviations used in this paper: FRET, Förster resonance energy transfer; Oxo-M, oxotremorine M; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PM, plasma membrane; PS, phosphatidylserine; SCG, superior cervical ganglion; SOCE, store-operated calcium entry; TIRF, total internal reflection fluorescence; UHP LC/MS, ultrahigh-performance liquid chromatography coupled with mass spectrometry; VSP, voltage-sensing 5-phosphatase.

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P 2 pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PH PLCδ1 domains as real-time indicators of PM PI(4,5)P 2 , and translocation of PH OSH2×2 , and PH OSH1 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)P 2 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)P 2 . Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P 2 . The decline of PI(4,5)P 2 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)P 2 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)P 2 when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI (4,5)P 2 production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.phosphoinositides | wortmannin | pleckstrin homology domain T his paper concerns the dynamics of cellular pools of phosphoinositides, a family of phospholipids located on the cytoplasmic leaflet of cellular membranes, that maintain cell structure, cell motility, membrane identity, and membrane trafficking; they also play key roles in signal transduction (1). Phosphatidylinositol (PI) can be phosphorylated at three positions to generate seven additional species. The subcellular localization of each phosphoinositide is tightly governed by the concurrent presence of lipid kinases and lipid phosphatases (2, 3), giving each membrane within the cell a unique and dynamic phosphoinositide signature (4). Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] is localized to the inner leaflet of the plasma membrane (PM) and is the major substrate of phospholipase C (PLC). As a consequence, PI(4,5)P 2 levels are dynamically regulated by G q -coupled receptors activating PLC. The activity of lipid kinases and phosphatases also can be modulated by signaling; for example, a PI 4-kinase, when associated with neuronal calcium sensor-1, is accelerated in response to elevated calcium that occurs with PI(4,5)P 2 cleavage (5). In addition, transient apposition between organelles can alter phosphoinositide levels by presenting membrane-bound phosphatases in trans. For example, the endoplasmic reticulum (ER) can make contacts with the...

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 ␣ 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 G␣ subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of G␣. Combining our data with a predictive Class A GPCR/G␣ model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of G␣. Such an interaction could promote opening of switch II of G␣ to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.A diverse network of signaling pathways have evolved within the hypothalamic-pituitary-gonadal axis to ensure precise neuroendocrine regulation of reproductive function in mammals (1). An essential feature of this physiological system is the pulsatile release of gonadotropin-releasing hormone from hypothalamic neurons, which subsequently initiates follicle-stimulating hormone and luteinizing hormone release from the pituitary and ultimately impinges on the gonads to elicit sex steroid secretion (2). Together, the components of the hypothalamic-pituitary-gonadal axis function with precise temporal and spatial accuracy to regulate the development and maintenance of proper reproductive function, including puberty onset and the estrous cycle (3). Thus, functional mutations in key elements of this critical physiological system can result in the development of various reproductive disorders. For example, idiopathic hypogonadotropic hypogonadism (IHH), 2 which is characterized by delayed or absent puberty, immature reproductive organs, low levels of sex steroids and infertility, is commonly associated with loss-of-function mutations in the gonadotropin-releasing hormone receptor (4, 5). More recently, IHH-causing mutations were identified in a relatively uncharacterized orphan G-protein-coupled recep...