RT Journal Article
SR Electronic
T1 Cannabinoid Receptor Interacting Protein (CRIP) 1a competition with β-arrestin for CB1 receptor binding sites
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.116.104638
DO 10.1124/mol.116.104638
A1 Lawrence C. Blume
A1 Theresa Patten
A1 Khalil Eldeeb
A1 Sandra Leone-Kabler
A1 Alexander A. Ilyasov
A1 Bradley M. Keegan
A1 Jeremy E. O'Neal
A1 Caroline E. Bass
A1 Roy R. Hantgan
A1 W. Todd Lowther
A1 Dana E. Selley
A1 Allyn C. Howlett
YR 2016
UL http://molpharm.aspetjournals.org/content/early/2016/11/28/mol.116.104638.abstract
AB 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.