The CB 1 cannabinoid receptor is a G-protein coupled receptor that has important physiological roles in synaptic plasticity, analgesia, appetite, and neuroprotection. We report the discovery of two structurally related CB 1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB 1 . CRIP1a and CRIP1b are generated by alternative splicing of a gene located on chromosome 2 in humans, and orthologs of CRIP1a occur throughout the vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB 1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB 1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB 1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB 1 -mediated tonic inhibition of voltage-gated Ca 2ϩ channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB 1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB 1 activity has therapeutic potential (e.g., chronic pain, obesity, and epilepsy).G protein-coupled receptors (GPCRs) provide a wide range of signaling capabilities to regulate the activity of downstream cellular targets. To signal efficiently, cells must be able to dynamically control the activity of GPCRs. Although some regulatory pathways, such as desensitization and internalization mediated by ␤-arrestin (Benovic et al., 1986), are applicable to most GPCRs, specialized means of regulation for particular GPCRs have been identified. Because many GPCRs have been shown to have spontaneous basal activity, ancillary proteins that interact with GPCRs may prove to be specific modulators of this activity. A prominent protein-protein interaction site studied on GPCRs is the C-terminal tail; G-protein binding and post-translational modifications occur in this region in many GPCRs. The profound sequence variety of C-terminal tails provides a means for selectivity in G-protein interactions as well as diversity in receptor trafficking. The G-protein-coupled receptor-associated sorting protein GASP1 interacts with the C-terminal tail of many GPCRs, including CB 1 , resulting in down-regulation and degradation (Martini et al., 2007). The adaptor protein FAN is also able to interact with the CB 1 receptor

Summary Background Nociceptive sensitization is a tissue damage response whereby sensory neurons near damaged tissue enhance their responsiveness to external stimuli. This sensitization manifests as allodynia (aversive withdrawal to previously nonnoxious stimuli) and/or hyperalgesia (exaggerated responsiveness to noxious stimuli). Although some factors mediating nociceptive sensitization are known, inadequacies of current analgesic drugs have prompted a search for additional targets. Results Here we use a Drosophila model of thermal nociceptive sensitization to show that Hedgehog (Hh) signaling is required for both thermal allodynia and hyperalgesia following ultraviolet irradiation (UV)-induced tissue damage. Sensitization does not appear to result from developmental changes in the differentiation or arborization of nociceptive sensory neurons. Genetic analysis shows that Hh signaling acts in parallel to tumor necrosis factor (TNF) signaling to mediate allodynia and that distinct transient receptor potential (TRP) channels mediate allodynia and hyperalgesia downstream of these pathways. We also demonstrate a role for Hh in analgesic signaling in mammals. Intrathecal or peripheral administration of cyclopamine (CP), a specific inhibitor of Sonic Hedgehog signaling, blocked the development of analgesic tolerance to morphine (MS) or morphine antinociception in standard assays of inflammatory pain in rats and synergistically augmented and sustained morphine analgesia in assays of neuropathic pain. Conclusions We demonstrate a novel physiological role for Hh signaling, which has not previously been implicated in nociception. Our results also identify new potential therapeutic targets for pain treatment.

The biarylpyrazole, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) has been shown to act as an inverse agonist/antagonist at the cannabinoid CB1 receptor. Our previous mutant cycle study suggested that K3.28(192) is involved in a direct interaction with the C-3 substituent of 1 in wild-type (WT) CB1.(1) However, these results did not establish what part of the C-3 substituent of 1 is involved in the K3.28(192) hydrogen bond, the carboxamide oxygen or the piperidine nitrogen. Furthermore, our previous calcium channel assay results for 5-(4- chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole (VCHSR; 2) (an analogue of 1 that lacks hydrogen-bonding capability in its C-3 substituent) showed that this compound acts as a neutral antagonist, a result that is in contrast to 1, which acts as an inverse agonist in this same assay.(1) These results suggested a relationship between biarylpyrazole interaction with K3.28(192) at CB1 and inverse agonism, but these results were for a single pair of compounds (1 and 2). The work presented here was designed to test two hypotheses derived from our modeling and mutant cycle results. The hypotheses are as follows: (1) it is the carboxamide oxygen of the C-3 substituent of 1 that interacts directly with K3.28(192) and (2) the interaction with K3.28(192) is crucial for the production of inverse agonism for biarylpyrazoles such as 1. To determine whether the carboxamide oxygen or the piperidine nitrogen of the C-3 substituent may be the interaction site for K3.28(192), we designed, synthesized, and evaluated a new set of analogues of 1 (3-6, Chart 1) in which modifications only to the C-3 substituent of 1 have been made. In each case, the modifications that were made preserved the geometry of this substituent in 1. The absence of the piperidine nitrogen was not found to affect affinity, whereas the absence of the carboxamide oxygen resulted in a reduction in affinity. CB1 docking studies in an inactive state model of CB1 resulted in the trend, 3,1<5,4<2<6 for ligand/CB1 interaction energies. This trend was consistent with the trend in WT CB1 Ki values versus [3H]CP55,940 reported here. In calcium channel assays, all analogues with carboxamide oxygens (1, 3, and 4) were found to be inverse agonists, whereas those that lacked this group (2, 5, and 6) were found to be neutral antagonists. Taken together, these results support the hypothesis that it is the carboxamide oxygen of the C-3 substituent of 1 that engages in a hydrogen bond with K3.28(192) in WT CB1. Furthermore, functional results for 1-6 support the hypothesis that the interaction of 1 with K3.28(192) may be key to its inverse agonism.

The intracellular C-terminal helix 8 (H8) of the CB 1 cannabinoid receptor deviates from the highly conserved NPXXY(X) 5,6 F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB 1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB 2 sequence. The two mutant receptors differed from wild type ( 2؉ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to G␣ i3 but not G␣ 0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by G␣ i3 . Furthermore, G␣ i3 but not G␣ 0A enhanced basal facilitation ratio, suggesting that G␣ i3 is responsible for CB 1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with G␣ i1 or G␣ i2 but not with G␣ i3 . Molecular dynamics simulations of WT CB 1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X) 5,6 L contributes to maximal activity of the CB 1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.