title: Cannabinoid receptor interacting protein suppresses agonist-driven CB1receptor internalization and regulates receptor replenishment in an agonist-biased manner.
abstract: Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1receptor (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.
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.
authors: Lawrence C
authors: LC
authors: Blume
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.
authors: Sandra
authors: S
authors: Leone-Kabler
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.
authors: Deborah J
authors: DJ
authors: Luessen
authors: Department of Biology, Wake Forest University, Winston-Salem, North Carolina, USA.|Center for Molecular Signaling, Wake Forest University, Winston-Salem, North Carolina, USA.
authors: Glen S
authors: GS
authors: Marrs
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.
authors: Erica
authors: E
authors: Lyons
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.
authors: Caroline E
authors: CE
authors: Bass
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.|Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA.
authors: Rong
authors: R
authors: Chen
authors: Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA.
authors: Dana E
authors: DE
authors: Selley
authors: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA. ahowlett@wakehealth.edu.|Center for Molecular Signaling, Wake Forest University, Winston-Salem, North Carolina, USA. ahowlett@wakehealth.edu.
authors: Allyn C
authors: AC
authors: Howlett
mesh_terms: D000818:Animals; D048588:Benzoxazines / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003511:Cyclohexanols / Q000494:pharmacology; D063388:Endocannabinoids / Q000502:physiology; D018628:Gene Dosage; D055785:Gene Knockdown Techniques; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D021381:Protein Transport; D011720:Pyrazoles / Q000494:pharmacology; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects; D000077285:Rimonabant; D015398:Signal Transduction / Q000235:genetics
chemical_list: D048588:Benzoxazines; C000602666:CRIP1a protein, mouse; D002352:Carrier Proteins; D003511:Cyclohexanols; D063388:Endocannabinoids; D009025:Morpholines; D009281:Naphthalenes; D010880:Piperidines; D011720:Pyrazoles; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1; D011956:Receptors, Cell Surface; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000077285:Rimonabant
keywords: CB1 cannabinoid receptor; CP55940; WIN55212-2; cannabinoid receptor interacting protein; receptor trafficking; rimonabant