TY - JOUR T1 - Cannabinoid Receptor Interacting Protein (CRIP) 1a competition with β-arrestin for CB1 receptor binding sites JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.116.104638 SP - mol.116.104638 AU - Lawrence C. Blume AU - Theresa Patten AU - Khalil Eldeeb AU - Sandra Leone-Kabler AU - Alexander A. Ilyasov AU - Bradley M. Keegan AU - Jeremy E. O'Neal AU - Caroline E. Bass AU - Roy R. Hantgan AU - W. Todd Lowther AU - Dana E. Selley AU - Allyn C. Howlett Y1 - 2016/01/01 UR - http://molpharm.aspetjournals.org/content/early/2016/11/28/mol.116.104638.abstract N2 - Cannabinoid Receptor Interacting Protein1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminus-associated protein that alters CB1R interactions with G-proteins (Blume et al.,2015; Smith et al.,2015). 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-terminal. Co-immunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to co-immunoprecipitate 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 over-expression attenuated CP55940-mediated GFP-CB1R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knock-down 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. Co-immunoprecipitation 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-arrestin1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB1R domains which could affect agonist-driven, β-arrestin-mediated internalization of the CB1R. ER -