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...

Sequence-specific nucleated protein aggregation is closely linked to the pathogenesis of most neurodegenerative diseases and constitutes the molecular basis of prion formation 1 . Here we report that fibrillar polyglutamine peptide aggregates can be internalized by mammalian cells in culture where they gain access to the cytosolic compartment and become co-sequestered in aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. Remarkably, these internalized fibrillar aggregates are able to selectively recruit soluble cytoplasmic proteins with which they share homologous, but not heterologous amyloidogenic sequences and to confer a heritable phenotype upon cells expressing the homologous amyloidogenic protein from a chromosomal locus.Genetic expansion of CAG triplets in protein-coding regions of otherwise unrelated genes is the underlying cause of a family of dominantly inherited neurodegenerative diseases including Huntington disease (HD) and spinocerebellar ataxias 2 . The tracts of polyglutamine (polyQ) homopolymers (Q ≥ 40) encoded by these expanded CAG triplets cause the normally soluble protein products of these genes, or fragments thereof, to form cytotoxic protein aggregates 2 . CAG expansion diseases therefore, belong to a much larger family of protein "conformational diseases," including systemic and organ-specific amyloidosis, Alzheimer's disease and prion encephalopathy. Pathogenesis in these diseases is tightly linked to the formation of high molecular weight, fibrillar, β-sheet rich, insoluble protein aggregates, termed "amyloid," that accumulate in characteristic sites either inside or outside of the cell 1, 3 .In amyloidosis, insoluble protein fibrils derived from normally soluble secreted proteins are deposited in the extracellular milieu causing damage to surrounding viscera, blood vessel walls and connective tissue 4 . Whether organ damage is a consequence of tissue disruption or obstruction due to the sheer mass of deposited protein, as in the case of systemic amyloidosis 4 , or to an intrinsic cytotoxicity of amyloids or their oligomeric precursors, as in the case of neuropathic amyloidosis 5 , remains a critical but unresolved question. In contrast to amyloidosis, most neurodegenerative diseases are caused by alterations in the conformation and oligomeric state of normally well-behaved intracellular proteins that, in diseased states, accumulate within cytoplasmic or nuclear inclusion bodies 6 . Emerging evidence suggests that oligomeric precursors to these large assemblies are cytotoxic and directly impair crucial Many extracellular amyloids and amyloid precursors, including those associated with systemic amyloidosis, neurodegenerative disease, and even those not associated with disease 7 , can be taken-up by a wide variety of cell types including macrophages, neurons, fibroblasts, and epithelial cells [7][8][9][10] . This uptake is reported to occur via phagocytic or endocytic processes that result in delivery to lysosomes which may suppress...

Central nervous system responses to cannabis are primarily mediated by CB 1 receptors, which couple preferentially to Gi/o G proteins. Here, we used calcium photometry to monitor the effect of CB1 activation on intracellular calcium concentration. Perfusion with 5 M CB1 aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB1 and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB1 antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including ⌬ 9 -tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G i/o, because it was present in pertussis toxintreated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by Gq G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative Gq or treated with the phospholipase C inhibitors U73122 and ET-18-OCH3 and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco͞endoplasmic reticulum Ca 2؉ pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1-diheptyl-4,4-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB1 receptors in a conformation that enables Gq signaling, thus shifting the G protein specificity of the receptor.aminoalkylindole ͉ inositol phosphate ͉ neurons ͉ phospholipase C T he CB 1 cannabinoid receptor (CB 1 ) mediates the majority of the psychotropic and behavioral effects of cannabis (1, 2). CB 1 is a member of the heptahelical G protein-coupled receptor (GPCR) superfamily (1). It couples via pertussis toxin (PTX)-sensitive G i/o G proteins to inhibit adenylyl cyclase and L-, N-, and P͞Q-type calcium channels and to activate potassium channels and mitogen-activated protein kinase (2). Rarely, CB 1 has also been found to couple to phospholipase C (PLC) in a PTX-sensitive manner involving the ␤␥ subunits from G i/o (3, 4).Several distinct agonists activate CB 1 . The classical cannabinoids are tricyclic dibenzopyran compounds. The prototype of this group is ⌬ 9 -tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabis. THC is a low-affinity partial agonist for CB 1 (2), whereas other synthetic classical compounds such as HU-210 are both more potent and efficacious (2). Nonclassical cannabinoids lack the dihydropyran ring yet maintain some similarity to THC's stereochemistry. The best known, CP55,940 (CP), is a potent and effic...

Background: Polyglutamine aggregates can be internalized by mammalian cells and gain access to the cytoplasmic compartment, but the properties of the aggregates and cell surface that mediate these processes are unknown. Results: Introduction of net negative charge and disruption of fibrillar structure greatly reduced the capacity of polyglutamine aggregates to bind and be internalized by mammalian cells. Conclusion: Aggregate uptake is influenced by the structure and net charge of aggregates and is mediated by two classes of binding sites on the cell surface. Significance: Elucidating how protein aggregates are internalized by cells is important for understanding the pathogenesis of many neurodegenerative disorders.

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