CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses.
SUMMARY The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues to identify in vivo substrates and to understand ABHD6's role in energy metabolism. Here we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high fat diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as novel therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CBR) distal C-terminal-associated protein that alters CBR interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CBR interactions with β-arrestin proteins that interact with the CBR at the C-terminus. Coimmunoprecipitation studies indicated that CBR associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CBR, which we hypothesized could attenuate the action of β-arrestin to mediate CBR internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CBRs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CBR. CRIP1a overexpression attenuated CP55940-mediated GFP-CBR as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CBR C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CBR distal C-terminus 14-mer peptide reduced CBR-CRIP1a association. Coimmunoprecipitation of CBR protein complexes demonstrated that central or distal C-terminal peptides competed for the CBR association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CBR domains that could affect agonist-driven, β-arrestin-mediated internalization of the CBR.
CB1 cannabinoid receptors (CB1R) are one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and regulate diverse neuronal functions. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCRs. The Cannabinoid Receptor Interacting Protein 1a (CRIP1a) binds to the distal carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function [1]. The mechanisms by which CRIP1a regulates CB1R activity have not yet been identified; therefore the focus of this investigation is to examine the cellular effects of CRIP1a on CB1R signaling using neuronal N18TG2 cells stably transfected with CRIP1a over-expressing and CRIP1a knockdown constructs. Modulation of endogenous CRIP1a expression did not alter total levels of CB1R, ERK, or forskolin-activated adenylyl cyclase activity. When compared to WT cells, CRIP1a over-expression reduced basal phosphoERK levels, whereas depletion of CRIP1a augmented basal phosphoERK levels. Stimulation of phosphoERK by the CB1R agonists WIN55212-2, CP55940 or methanandamide was unaltered in CRIP1a over-expressing clones compared with WT. However, CRIP1a knockdown clones exhibited enhanced ERK phosphorylation efficacy in response to CP55940. In addition, CRIP1a knockdown clones displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation. CB1R-mediated Gi3 and Go activation by CP99540 was attenuated by CRIP1a over-expression, but robustly enhanced in cells depleted of CRIP1a. Conversely, CP55940-mediated Gi1 and Gi2 activation was significant enhanced in cells over-expressing CRIP1a, but not in cells deficient of CRIP1a. These studies suggest a mechanism by which endogenous levels of CRIP1a modulate CB1R-mediated signal transduction by facilitating a Gi/o-protein subtype preference for Gi1 and Gi2, accompanied by an overall suppression of G-protein-mediated signaling in neuronal cells.
Cannabinoid CB 1 receptors (CB 1 Rs) 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 CB 1 R C-terminus and can attenuate constitutive CB 1 R-mediated inhibition of Ca 21 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 CB 1 Rmediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP 1a in human embryonic kidney (HEK)-293 cells stably expressing CB 1 Rs (CB 1 -HEK), or in N18TG2 cells endogenously expressing CB 1 Rs, decreased CB 1 Rmediated G-protein activation (measured by agonist-stimulated [ activation. These effects were not attributable to differences in CB 1 R expression or endocannabinoid tone because CB 1 R levels did not differ between cell lines varying in CRIP 1a expression, and endocannabinoid levels were undetectable (CB 1 -HEK) or unchanged (N18TG2) by CRIP 1a overexpression. In CB 1 -HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB 1 Rs and desensitized agonist-stimulated [ 35 S]GTPgS binding. CRIP 1a overexpression attenuated CB 1 R downregulation without altering CB 1 R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP 1a overexpression attenuated both depolarizationinduced 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 CB 1 R activity and demonstrate that CRIP 1a can also inhibit agonist-stimulated CB 1 R signaling and downregulation of CB 1 Rs. Thus, CRIP 1a appears to act as a broad negative regulator of CB 1 R function.
Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1R) and D2 dopamine (D2R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1R or D2R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1R and D2R knockdown reduced striatal dopaminergic-stimulated [35S] GTPγS binding, and D2R knockdown reduced pallidal WIN55212-2-stimulated [35S]GTPγS binding. Decreased D2R and CB1R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1Rs or D2Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [35S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1R or D2R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminus-associated protein that modulates CB1R signaling via G proteins, and CB1R down-regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716–726; Smith et al. [2015] Mol. Pharmacol., 87, 747–765). In this study, we determined the involvement of CRIP1a in CB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB1R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30–120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB1Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)-mediated increase in cell surface CB1Rs, which is postulated to be as a result of rimonabant effects on ‘non-agonist-driven’ internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface.
Recent studies have used conditional knockout mice to selectively delete the D2 autoreceptor; however, these approaches result in global deletion of D2 autoreceptors early in development. The present study takes a different approach using RNA interference (RNAi) to knockdown the expression of the D2 receptors (D2R) in the substantia nigra (SN), including dopaminergic neurons, which project primarily to the dorsal striatum (dStr) in adult rats. This approach restricts the knockdown primarily to nigrostriatal pathways, leaving mesolimbic D2 autoreceptors intact. Analyses of dopamine (DA) kinetics in the dStr reveal a decrease in DA transporter (DAT) function in the knockdown rats, an effect not observed in D2 autoreceptor knockout mouse models. SN D2 knockdown rats exhibit a behavioral phenotype characterized by persistent enhancement of locomotor activity in a familiar open field, reduced locomotor responsiveness to high doses of cocaine and the ability to overcome haloperidol-induced immobility on the bar test. Together these results demonstrate that presynaptic D2R can be depleted from specific neuronal populations and implicates nigrostriatal D2R in different behavioral responses to psychotropic drugs.
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