Supplementary Materials Data Supplement supp_87_4_747__index. [35S]GTPgene, which contains four exons: 1,

Supplementary Materials Data Supplement supp_87_4_747__index. [35S]GTPgene, which contains four exons: 1, 2, 3a, and 3b. Alternate splicing produces transcripts comprising exons 1, 2, and 3a (CRIP1a) or 1, 2, and 3b (CRIP1b). NSC 23766 inhibitor CRIP1a homologs are found throughout vertebrates, whereas CRIP1b appears to be limited to primates (Niehaus et al., 2007). The search for CB1R C-terminalCinteracting proteins was initiated because this region exhibited autoinhibition of constitutive (agonist-independent) CB1R activity, which was relieved by truncation of the distal C-terminus of the receptor (Nie and Lewis, 2001a,b). Indeed, electrophysiological recordings in superior cervical ganglion (SCG) neurons showed that expression of CRIP1a, but not CRIP1b, attenuated constitutive CB1-mediated inhibition of calcium channels, revealed by elimination of the inverse agonist activity of rimonabant (SR141716A). However, coexpression of CRIP1a and CB1Rs did not alter agonist-induced inhibition of calcium currents or CB1R expression levels (Niehaus et al., 2007), suggesting that CRIP1a inhibits constitutive CB1R activity. CRIP1a is usually highly expressed in the brain (Niehaus et al., 2007), and some reports suggest that CRIP1a is usually regulated by seizure activity. Sclerotic hippocampi from epileptic patients exhibited reduced expression of mRNA for both CRIP1a and CB1R (Ludanyi et al., 2008). In contrast, CRIP1a mRNA was elevated in rat hippocampus and cortex following kainic acidCinduced seizures (Bojnik et al., 2012). These findings suggest CRIP1a involvement in modulating CB1R function in the pathogenesis or neuroadaptive response to epilepsy. Furthermore, CRIP1a expression inhibited the neuroprotective effects of a cannabinoid agonist and conferred a neuroprotective effect on an antagonist, in a cultured neuronal model of glutamate excitotoxicity (Stauffer et al., 2011). To date, evidence supports functional interactions between CRIP1a and CB1R in striatal GABAergic medium spiny neurons (Blume et al., 2013), glutamatergic hippocampal neurons (Ludanyi et al., 2008), and retinal presynaptic terminals (Hu et al., 2010). In addition, the gene is usually hypermethylated in certain colorectal cancers (Lind et al., 2011; Oster et al., 2011), further suggesting potentially important functions of CRIP1a in multiple physiologic systems. Despite the potential significance of CRIP1a as a novel player in the endocannabinoid system, relatively little is known about its function. The present study decided the effects of CRIP1a on constitutive and agonist-stimulated G-protein activation in CB1R-expressing cells. Because CRIP1a binds to the CB1R C-terminus, which interacts with regulatory proteins that mediate CB1R desensitization and downregulation, the effects of CRIP1a on prolonged agonist-induced adaptation in CB1R expression and signaling were also examined. To examine colocalization of CRIP1a with CB1Rs in a defined neuronal populace in the CNS, colabeling studies were conducted in the cerebellum because both proteins are highly expressed in this NSC 23766 inhibitor region (Herkenham et al., 1991; Niehaus et al., 2007) and it plays a major role in cannabinoid dependence (Tzavara et al., 2000). Finally, to investigate the effects of NSC 23766 inhibitor CRIP1a on endocannabinoid function, its influence on depolarization-induced suppression of excitation (DSE) was examined in autaptic hippocampal neurons. Materials and Methods Chemicals [35S]GTPvector (hCB1-HEK-CRIP1a) (Niehaus et al., 2007) were cultured in the same media with the addition of 0.1 mg/ml zeocin. Stable CRIP1a-overexpression and -knockdown N18TG2 cell clones were generated by transfecting (Lipofectamine 2000; Invitrogen, Carlsbad, CA) N18TG2 cells with either a pcDNA3.1-CRIP1a mouse cDNA plasmid for overexpression, or two different pRNATin-H1.2 small-interfering RNA (siRNA)-CRIP1a vectors for knockdown. The GenScript siRNA target finder program NSC 23766 inhibitor (GenScript, Piscataway, NJ) was used to select CRIP1a TH siRNA-target sequences. CRIP1a N18TG2 cell lines were generated by isolating and expanding G418-resistent single colonies in selection NSC 23766 inhibitor media made up of 600 for 10 minutes to remove media. Cells were homogenized in ice-cold 50 mM Tris-HCl, 3 mM MgCl2, and 1 mM EGTA, pH 7.4 (membrane buffer), and centrifuged at 50,000for 10 minutes. The producing pellets were homogenized in 50 mM.