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

Cannabinoid receptor interacting protein 1a (CRIP1a) is aCB1receptor (CB1R) distal C‐terminus‐associated protein that modulatesCB1R signaling via G proteins, andCB1R down‐regulation but not desensitization (Blumeet al. [2015]Cell Signal., 27, 716–726; Smithet al. [2015]Mol. Pharmacol., 87, 747–765). In this study, we determined the involvement ofCRIP1a inCB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surfaceCB1Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses bothCB1R andCRIP1a, and exhibits a well‐characterized endocannabinoid signaling system. We developed stableCRIP1a‐over‐expressing andCRIP1a‐siRNA‐silenced knockdown clones to investigate gene dose effects ofCRIP1a onCB1R plasma membrane expression. Results indicate thatCP55940 or WIN55212‐2 (10 nM, 5 min) reduced cell surfaceCB1R by a dynamin‐ and clathrin‐dependent process, and this was attenuated byCRIP1a over‐expression.CP55940‐mediated cell surfaceCB1R 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 surfaceCB1Rs recovered only inCRIP1a knockdown cells. Changes inCRIP1a expression levels did not affect a transient rimonabant (10 nM)‐mediated increase in cell surfaceCB1Rs, which is postulated to be as a result of rimonabant effects on ‘non‐agonist‐driven’ internalization. These studies demonstrate a novel role forCRIP1a in agonist‐drivenCB1R 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 ofde novosynthesized receptor to the cell surface.