CB1 cannabinoid receptor location and CRIP1a

Abstract

CB 1 cannabinoid receptor (CB 1 R) is a Gi/o linked GPCR that influences psychological processes such as mood and memory. Cannabinoid Receptor Interacting Protein (CRIP1a) has been shown to interact with the C‐terminal tail of the CB 1 R, leading to the hypothesis that CRIP1a could influence receptor localization and trafficking. The goal of this study was to identify the role of CRIP1a in CB 1 R localization using N18TG2 wild type (WT) and CRIP1a over‐expressing neuroblastoma cell clones as a model system. Cells were transfected with green fluorescent protein‐tagged CB 1 receptors (GFP‐CB 1 R). GFP‐CB 1 R and CRIP1a were imaged using confocal microscopy, and the localization was identified using nuclear (HCS nuclear mask) and early endosome (Cell‐Lights) molecular probes. N18TG2 WT cells displayed CRIP1a uniformly in the cell cytosol. At basal conditions, the GFP‐CB 1 Rs in WT cells are primarily expressed on the plasma membrane surface. On cell Western studies using an N‐terminally‐directed CB 1 antibody, revealed that stable over‐expression of CRIP1a reduced the density of CB 1 R's on the plasma membrane, when compared to WT cells. During agonist (WIN55212‐2)‐challenge, internalization of CB 1 receptors occurs in WT cells, but not in CRIP1a‐overexpressing clones. Upon exposure to the CB 1 antagonist rimonabant, the density of cell‐surface CB 1 receptors increased in both WT as well as CRIP1a‐overexpressing cells. These studies suggest the CRIP1a influences CB1R trafficking and localization during basal and agonist conditions, and provides a mechanisms for regulating CB1 receptor availability at the plasma membrane. Supported by: GM‐ GM064249 , RO1‐DA03690, R21‐ DA025321