Cannabinoid receptor interacting protein suppresses agonist‐driven CB₁ receptor internalization and regulates receptor replenishment in an agonist‐biased manner

Abstract

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.

Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites

“…Thus, we cannot rule out the possibility that if CRIP1a bound to the CB 1 R precludes a CB 1 R-Gi3 or Go complex, then a subsequent phosphorylation and interactions with b-arrestin might not be initiated. In studies of the agonist-mediated depletion of cell surface CB 1 R, we found that CRIP1a overexpression prevented CB 1 R internalization (Blume et al, 2016). This finding is completely consistent with a competition by CRIP1a for a binding site for b-arrestin on the CB 1 R C-terminal and the amelioration of the b-arrestinmediated internalization processes.…”

“…2B). The clustering of GFP-CB 1 R in the CRIP1a XS cells before agonist stimulation is a phenomenon that will require greater investigation, because data from our studies of exposure to agonist ligands suggest that CRIP1a could play an inhibitory role in trafficking of the CB 1 R to the plasma membrane (Blume et al, 2016). Exposure to CP55940 (10 nM, 15 minutes) resulted in a pronounced redistribution of GFP-CB 1 R fluorescence into discrete punctate aggregates along the membrane surface in WT and empty-vector control cells.…”

“…CB 1 R cell surface density and internalization were quantified using a 96-well format "On-cell-Western" immunocytochemistry assay, as previously reported (Miller, 2004;Blume et al, 2015Blume et al, , 2016Smith et al, 2015). N18TG2 cells have been reported to synthesize the full agonist endocannabinoid 2-AG (Bisogno et al, 1997).…”

CRIP1a inhibits endocytosis of G‐protein coupled receptors activated by endocannabinoids and glutamate by a common molecular mechanism

“…), which can be inhibited by CRIP1a in the N18TG2 cell line (Blume et al . ,b). To analyze in detail underlying molecular mechanisms using a previously established internalization assay in HEK293 cells (Fig.…”

In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) – CB1 cannabinoid receptor

“…CRIP1a plays a role in the control of agonist-driven CB 1 R cell surface regulation by competing with β-arrestins and thereby attenuating agonist-mediated internalization[14,15]. CRIP1a over-expression attenuated the agonist-induced redistribution of endogenous β-arrestin to punctae aggregates and subsequent loss of cell surface CB 1 receptors.…”