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

Abstract

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CBR) distal C-terminal-associated protein that alters CBR interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CBR interactions with β-arrestin proteins that interact with the CBR at the C-terminus. Coimmunoprecipitation studies indicated that CBR 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 CBR, which we hypothesized could attenuate the action of β-arrestin to mediate CBR internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CBRs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CBR. CRIP1a overexpression attenuated CP55940-mediated GFP-CBR as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CBR 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 CBR distal C-terminus 14-mer peptide reduced CBR-CRIP1a association. Coimmunoprecipitation of CBR protein complexes demonstrated that central or distal C-terminal peptides competed for the CBR 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 CBR domains that could affect agonist-driven, β-arrestin-mediated internalization of the CBR.

In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) – CB1 cannabinoid receptor

“…The function of CRIP1a has been clearly established in CB 1 receptor selection of Gi/o protein preference and signaling, and competition for β-arrestin to attenuate CB 1 receptor internalization[14], whereas the function of CRIP1b is not known [11]. Exogenous CRIP1a reversed CB 1 receptor-mediated tonic inhibition of Ca 2+ currents, whereas CRIP1b could not[11].…”

“…CB 1 receptor-mediated G i3 and G o activation by CP55940 was attenuated by CRIP1a over-expression, but robustly enhanced in CRIP1a-knockdown clones. Conversely, CP55940-mediated G i1 and G i2 activation was significant enhanced in cells over-expressing CRIP1a, but not inCRIP1a-knockdown clones [14]. These studies suggest that endogenous levels of CRIP1a modulate CB 1 receptor-mediated signal transduction by facilitating a G i/o subtype preference for G i1 and G i2 , accompanied by an overall suppression of G protein-mediated signaling in neuronal cells [12,13].…”

“…CRIP1a plays a role in the control of agonist-driven CB 1 R cell surface regulation by competing with β-arrestins and thereby attenuating agonist-mediated internalization[14,15]. CRIP1a over-expression attenuated the agonist-induced redistribution of endogenous β-arrestin to punctae aggregates and subsequent loss of cell surface CB 1 receptors.…”

“…CRIP1a over-expression attenuated the agonist-induced redistribution of endogenous β-arrestin to punctae aggregates and subsequent loss of cell surface CB 1 receptors. Conversely, CRIP1a knock-down augmented agonist-mediated β-arrestin redistribution to punctae[14]. Co-immunoprecipitation studies indicated thatCRIP1a competes with β-arrestin for binding to the CB 1 receptor, which attenuates the action of β-arrestin to mediate CB 1 R internalization[14].…”

“…Conversely, CRIP1a knock-down augmented agonist-mediated β-arrestin redistribution to punctae[14]. Co-immunoprecipitation studies indicated thatCRIP1a competes with β-arrestin for binding to the CB 1 receptor, which attenuates the action of β-arrestin to mediate CB 1 R internalization[14]. Peptides mimicking the CB 1 receptor C-terminus could pull-down CRIP1a from cell extracts and recombinant CRIP1a.…”