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

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CB R) distal C-terminus-associated protein that modulates CB R signaling via G proteins, and CB 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 CB R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB 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 CB R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB 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 CB 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 CB 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 CB 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.