Cannabinoid receptor interacting protein 1a competition with β-arrestin for CB1 receptor binding sites

Abstract

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 beta-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or b-arrestin, but CRIP1a and b-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and b-arrestin binding to the CB1R, which we hypothesized could attenuate the action of b-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB(1)Rs 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 beta-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented beta-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 b-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with beta-arrestin 2. Thus, CRIP1a can compete with beta-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, beta b-arrestin-mediated internalization of the CB1R.