Cannabinoid receptor interacting protein suppresses agonist-driven CB 1 receptor internalization and regulates receptor replenishment in an agonist-biased manner

J Neurochem. 2016 Nov;139(3):396-407. doi: 10.1111/jnc.13767. Epub 2016 Sep 26.

Abstract

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1 R) distal C-terminus-associated protein that modulates CB1 R signaling via G proteins, and CB1 R 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 CB1 R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1 Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1 R 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 CB1 R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB1 R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1 R 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 CB1 Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)-mediated increase in cell surface CB1 Rs, 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 CB1 R 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.

Keywords: CB1 cannabinoid receptor; CP55940; WIN55212-2; cannabinoid receptor interacting protein; receptor trafficking; rimonabant.

MeSH terms

  • Animals
  • Benzoxazines / pharmacology
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Cell Line
  • Cell Membrane / metabolism
  • Cyclohexanols / pharmacology
  • Endocannabinoids / physiology
  • Gene Dosage
  • Gene Knockdown Techniques
  • Mice
  • Morpholines / pharmacology
  • Naphthalenes / pharmacology
  • Piperidines / pharmacology
  • Protein Transport
  • Pyrazoles / pharmacology
  • RNA, Small Interfering
  • Receptor, Cannabinoid, CB1 / agonists*
  • Receptor, Cannabinoid, CB1 / genetics
  • Receptor, Cannabinoid, CB1 / metabolism*
  • Receptors, Cell Surface / drug effects
  • Rimonabant
  • Signal Transduction / genetics

Substances

  • Benzoxazines
  • CRIP1a protein, mouse
  • Carrier Proteins
  • Cyclohexanols
  • Endocannabinoids
  • Morpholines
  • Naphthalenes
  • Piperidines
  • Pyrazoles
  • RNA, Small Interfering
  • Receptor, Cannabinoid, CB1
  • Receptors, Cell Surface
  • (3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone
  • 3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol
  • Rimonabant