CB 1 Cannabinoid Receptor Activity Is Modulated by the Cannabinoid Receptor Interacting Protein CRIP 1 a

The CB1 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 CB1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB1. 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 CB1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB1-mediated tonic inhibition of voltage-gated Ca channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB1 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 CB1, resulting in down-regulation and degradation (Martini et al., 2007). The adaptor protein FAN is also able to interact with the CB1 receptor This work was supported by the National Institute on Drug Abuse (D.L.L, J.L.N., A.C.H., and D.E.S.), the Biotechnology and Biological Sciences Research Council (M.R.E.), and the Medical College of Georgia Research Institute (D.L.L). J.L.N., Y.L., K.T.W. and M.E. contributed equally to this work. 1 Current affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island. 2 Current affiliation: Department of Physiology & Biophysics, Baylor College of Medicine, Houston, Texas. 3 Current affiliation: Department of Anesthesiology, UT-MD Anderson Cancer Center, Houston, Texas. 4 Current affiliation: Department of Physiology and Pharmacology, Wake Forest University, Winston-Salem, North Carolina. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.039263. ABBREVIATIONS: GPCR, G protein-coupled receptor; CB1, cannabinoid receptor subtype-1; CRIP1a, cannabinoid receptor interacting protein subtype 1a; CRIP1b, cannabinoid receptor interacting protein subtype 1b; SCG, superior cervical ganglion; WIN 55,212-2, [2,3-dihydro-5-methyl3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone; SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; HEK, human embryonic kidney; CHO, Chinese hamster ovary; aa, amino acid(s); GST, glutathione transferase; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; BSA, bovine serum albumin; PDZ, postsynaptic density 95/disc-large/zona occludens; HA, hemagglutinin. 0026-895X/07/7206-1557–1566$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 6 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 39263/3282026 Mol Pharmacol 72:1557–1566, 2007 Printed in U.S.A. 1557 at A PE T Jornals on Jne 9, 2017 m oharm .aspeurnals.org D ow nladed from (Sánchez et al., 2001). Regulation of basal activity of GPCRs by accessory proteins binding to the C-terminal tail has been described for metabotropic glutamate receptors (mGluRs). One of the members of the Homer protein family, Homer 1a, uncovers constitutive basal activity of group I mGluRs by competing for mGluR1/5 binding with other Homer isoforms that normally prevent constitutive signaling (Ango et al., 2001). The CB1 cannabinoid receptor, a GPCR, is activated by -tetrahydrocannabinol (Howlett, 1985), the primary psychotropic component of marijuana, as well as endocannabinoids such as anandamide (Devane et al., 1992) and 2-arachidonyl glycerol (Mechoulam et al., 1995). Endocannabinoids act as retrograde messengers mediating CB1-dependent forms of short-term synaptic plasticity known as depolarization-induced suppression of inhibition or excitation (Diana and Marty, 2004) and longer-lasting forms of synaptic plasticity, such as long-term depression (Gerdeman and Lovinger, 2001; Robbe et al., 2002; Chevaleyre and Castillo, 2003; Sjöström et al., 2003; Azad et al., 2004). Extinction of aversive memories is dependent on the endocannabinoid system (Marsicano et al., 2002). The endocannabinoid system also mediates a neuroprotective effect in models of excitotoxicity (Shen and Thayer, 1998; Abood et al., 2001), ischemia (ParmentierBatteur et al., 2002) and seizure (Marsicano et al., 2003). The complexity of CB1 signaling is increased by the agonist-independent or ligand-free constitutive activity as measured by its reversal with the antagonist/inverse agonist SR141716 (Bouaboula et al., 1997). Application of SR141716 reverses the tonic inhibition of N-type voltage-gated Ca channels, resulting in an increase in the Ca current in superior cervical ganglion (SCG) neurons expressing CB1 receptors (Pan et al., 1998). Deletion of the CB1 C-terminal tail distal to the G-protein binding domain enhances the effect of SR141716. SR141716 produces a significantly larger increase in the Ca current in neurons expressing C-terminally truncated CB1 receptors (Nie and Lewis, 2001). Thus, deletion of the distal C-terminal region of CB1 results in enhanced tonic inhibition of Ca channels, suggesting that either this region constrains the receptor conformation or that accessory proteins binding to this region modulate CB1 activity. We report here the discovery of two cannabinoid receptor interacting proteins (CRIP), CRIP1a and CRIP1b, that interact with the distal C-terminal tail of CB1. CRIP1a is expressed in the brain and is found throughout vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB1 from rat brain and colocalizes with CB1 when heterologously expressed in neurons. Neither CRIP1a nor CRIP1b significantly alters the affinity of CB1 for the antagonist/inverse agonist SR141716. However, CRIP1a, but not CRIP1b, significantly attenuates tonic inhibition of voltage-gated Ca channels by CB1 receptors. Materials and Methods Yeast Two-Hybrid Screening. The Matchmaker Two-Hybrid System (Clontech, Mountain View, CA) was used to screen a human brain cDNA library (Clontech) using a bait protein corresponding to the C-terminal tail of CB1 (last 55 amino acids, 418–472, of human CB1, excluding the G-protein binding region 400–417). The positive clone with the highest -galactosidase activity as determined by filter-lift assay (i.e., CRIP1b) was isolated and cotransformed with bait cDNA (CB1 C-terminal tail) into yeast to confirm the interaction. PCR Screening of Rat Brain cDNA Library. A rat brain cDNA library was constructed using a GeneRacer kit (Invitrogen, Carlsbad, CA). Primers to homologous regions of CRIP1b were used in combination with GeneRacer kit primers to determine the full coding region of CRIP1a. Full-length CRIP1a was then cloned using primers just upstream from the start site (5 primer: CTT CCT CCC TGC CTG TCT CTG) and downstream from the stop site (3 primer: GCT GTT TAT GTT ATT ACC TCT). Accession numbers for CRIP1a (AY883936) and CRIP1b (AY144596) nucleotide sequences have been deposited into GenBank. In Vitro Binding Assay. The GST Gene Fusion System (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) was used to construct GST-CB1 (C-terminal tail) fusion proteins using the pGEX4T-1 vector. GST-CB1 was expressed in Escherichia coli, isolated with glutathione-Sepharose beads, and incubated with lysate containing CRIP1a or CRIP1b S-tag fusion proteins subcloned into pET44a( ) or pET30c, respectively. Eluted proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membrane. The S-tag [15 amino acids (aa)] was visualized by its interaction with ribonuclease S-protein conjugated to horseradish peroxidase (Novagen, San Diego, CA). Generation of CRIP1a Antibodies. Rabbits were immunized with a conjugate of thyroglobulin and a peptide comprising the last 17 amino acids of rat/mouse/human CRIP1a, followed by affinitypurification of antibodies from antisera using the immunizing