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.
- Received April 6, 2016.
- Accepted November 23, 2016.
This work was supported by National Institutes of Health National Institute on Drug Abuse [Grants R01-DA03690, R21-DA025321, K01-DA024763, T32-DA007246, and F31-DA032215, R03-DA035424, P50-DA006634]; National Institute on Alcoholism and Alcohol Abuse [Grant T32-AA007565]; National Cancer Institute [Grant P30-CA012197]; and National Institute of General Medical Sciences [Grants R25-GM064249, K12-GM102773]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
↵1 Current affiliation: Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104.
↵2 Current affiliation: Department of Pharmacology, Jerry M. Wallace School of Osteopathic Medicine, Campbell University, Buies Creek, NC 27506.
↵3 Current affiliation: Department of Natural Resources and Environmental Design, North Carolina A&T State University, Greensboro, NC 27411.
↵4 Current affiliation: Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14260.
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics