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.