Dana E. Selley

Cannabinoid CB₁ receptors (CB₁Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor–interacting protein 1a (CRIP_1a) binds to the CB₁R C-terminus and can attenuate constitutive CB₁R-mediated inhibition of Ca²⁺ channel activity. We now demonstrate cellular colocalization of CRIP_1a at neuronal elements in the CNS and show that CRIP_1a inhibits both constitutive and agonist-stimulated CB₁R-mediated guanine nucleotide–binding regulatory protein (G-protein) activity. Stable overexpression of CRIP_1a in human embryonic kidney (HEK)-293 cells stably expressing CB₁Rs (CB₁-HEK), or in N18TG2 cells endogenously expressing CB₁Rs, decreased CB₁R-mediated G-protein activation (measured by agonist-stimulated [³⁵S]GTPγS (guanylyl-5′-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB₁-HEK cells. Conversely, small-interfering RNA–mediated knockdown of CRIP_1a in N18TG2 cells enhanced CB₁R-mediated G-protein activation. These effects were not attributable to differences in CB₁R expression or endocannabinoid tone because CB₁R levels did not differ between cell lines varying in CRIP_1a expression, and endocannabinoid levels were undetectable (CB₁-HEK) or unchanged (N18TG2) by CRIP_1a overexpression. In CB₁-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB₁Rs and desensitized agonist-stimulated [³⁵S]GTPγS binding. CRIP_1a overexpression attenuated CB₁R downregulation without altering CB₁R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP_1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP_1a inhibits constitutive CB₁R activity and demonstrate that CRIP_1a can also inhibit agonist-stimulated CB₁R signaling and downregulation of CB₁Rs. Thus, CRIP_1a appears to act as a broad negative regulator of CB₁R function.

The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G‐protein‐coupled receptors (GPCRs), including CB₁and CB₂receptors. The CB₁receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB₁receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G‐proteins. These proteins, which include the GPCR kinases, β‐arrestins, GPCR‐associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor‐interacting proteins: CRIP_1a/b, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down‐regulation, signal transduction and constitutive activity of CB₁receptors. This review examines CB₁receptor‐interacting proteins, including heterotrimeric G‐proteins, but with particular emphasis on non‐G‐protein entities, that might comprise the CB₁receptosomal complex. The evidence for direct interaction with CB₁receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visithttp://dx.doi.org/10.1111/j.1476‐5381.2010.00831.x

Background and purpose: Chronic morphine administration produces tolerance in vivo and attenuation of μ opioid receptor (MOR)‐mediated G‐protein activation measured in vitro, but the relationship between these adaptations is not clear. The present study examined MOR‐mediated G‐protein activation in the CNS of mice with different levels of morphine tolerance.Experimental approach: Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR‐mediated behaviours. MOR function was measured using agonist‐stimulated [³⁵S]guanylyl‐5′‐O‐(γ‐thio)‐triphosphate ([³⁵S]GTPγS) and receptor binding throughout the CNS.Key results: Morphine pellet implantation produced 6‐12‐fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED₅₀ values between morphine‐treated and control groups. Pellet implantation plus supplemental injections produced 25‐50‐fold tolerance in these tests. In morphine pellet‐implanted mice, MOR‐stimulated [³⁵S]GTPγS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet‐implanted mice. In contrast, MOR‐stimulated [³⁵S]GTPγS binding was significantly decreased in most regions examined in morphine pellet+morphine injected mice, including nucleus accumbens, caudate‐putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord.Conclusions and implications: Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR‐mediated G‐protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS‐mediated effects of morphine.British Journal of Pharmacology (2007) 151, 1324–1333; doi:10.1038/sj.bjp.0707328

Sphingosine-1-phosphate (S1P) and cannabinoid receptors are G-protein-coupled receptors that mediate the effects of S1P and endocannabinoids, respectively. Cannabinoid receptors also mediate the effects of Δ⁹-tetrahydrocannabinol, the primary psychoactive ingredient in marijuana, whereas S1P receptors contribute to the immunosuppressant effects of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720). FTY720 is a sphingosine analog that can prevent renal graft rejections and suppress a variety of autoimmune disorders in animal models and clinical trials. We now report that both FTY720 and sphingosine interact with CB₁ but not CB₂ cannabinoid receptors. FTY720 and sphingosine inhibited the binding of the CB₁-selective antagonist [³H]N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide ([³H]SR141716A) and the cannabinoid agonist [³H](–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([³H]CP55,940) in a concentration-dependent manner in both CB₁-expressing cell lines and mouse cerebellum. However, these compounds did not significantly alter [³H]CP55,940 binding to CB₂ receptors. In G-protein activation assays, FTY720 and sphingosine inhibited the maximal stimulation of guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding by the cannabinoid agonist R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) in a concentration-dependent manner, and this antagonist effect was not mimicked by S1P. FTY720 and sphingosine also inhibited activation of extracellular signal-regulated kinases 1 and 2 and Akt by WIN55,212-2 in intact Chinese hamster ovary (CHO) cells expressing CB₁ receptors and attenuated WIN55,212-2-stimulated internalization of a fluorescence-tagged CB₁ receptor in CHO cells. Moreover, both FTY720 and sphingosine produced rightward shifts in the concentration-effect curves of cannabinoid agonists for G-protein activation, indicating that they act as competitive CB₁ antagonists. These results suggest that the CB₁ receptor could be a novel target of FTY720 and that sphingosine could be an endogenous CB₁ antagonist.