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

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB₁ receptor (CB₁R) distal C-terminal-associated protein that alters CB₁R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB₁R interactions with β-arrestin proteins that interact with the CB₁R at the C-terminus. Coimmunoprecipitation studies indicated that CB₁R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CB₁R, which we hypothesized could attenuate the action of β-arrestin to mediate CB₁R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB₁Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB₁R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB₁R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CB₁R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB₁R distal C-terminus 14-mer peptide reduced CB₁R-CRIP1a association. Coimmunoprecipitation of CB₁R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB₁R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB₁R domains that could affect agonist-driven, β-arrestin-mediated internalization of the CB₁R.