Binding between a Distal C-Terminus Fragment of Cannabinoid Receptor 1 and Arrestin-2

Abstract

Internalization of G-protein coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB15P454-473). Complexes formed between the peptide and human arrestin-2 (wt-arr21-418) were compared to those formed with a truncated arrestin-2 mutant (tr-arr21-382) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The penta-phosphopeptide CB15P454-473 adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a hepta-phosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh7P330-348), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB15P454-473 peptide binds to tr-arr21-382 with higher affinity than to the full-length wt-arr21-418. As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred nOe data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB15P454-473 peptide.

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

“…As described in the introduction, the binding between β-arr2 and CB 1 R as well as a role for β-arrestins in the desensitization of CB 1 R have been demonstrated by former studies (Bakshi et al, 2007;Daigle et al, 2008aDaigle et al, , 2008bJin et al, 1999;Kouznetsova et al, 2002;Nguyen et al, 2012;Singh et al, 2011). Therefore, the role of β-arr2 in CB 1 R endocytosis may seem to be a logical consequence of its recruitment to the receptor.…”

“…It has been demonstrated that serine and threonine residues at the C-terminus of CB 1 R are involved in its β-arr2 binding and agonist-induced endocytosis (Daigle et al, 2008b;Hsieh et al, 1999). In contrast to the various studies that clearly point to an interaction between CB 1 R and β-arr2 upon agonist stimulation, no direct data have been hitherto presented concerning the β-arr1 binding of CB 1 R. In some structural studies, the association of β-arr1 with a synthesized CB 1 R C-terminus has been shown (Bakshi et al, 2007;Singh et al, 2011), however, such binding has not been demonstrated with the intact CB 1 R in living cells.…”

Cannabinoid Receptor–Interacting Protein 1a Modulates CB1 Receptor Signaling and Regulation

“…For example, probable G-protein-coupled receptor kinase phosphorylation sites have been identified Jin et al, 1999;Daigle et al, 2008) and isolated fragments of the CB 1 R C-terminus can bind to b-arrestins (Singh et al, 2011;Bakshi et al, 2007) and GASP1 (Martini et al, 2007). Prolonged agonist exposure downregulates CB 1 Rs expressed in HEK-293 (Shapira et al, 2003) but not N18TG2 cells (McIntosh et al, 1998).…”

Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex

“…We also sampled side‐chain conformation of the CB1 Ct peptide using the rotamer database and obtained the best combination of side‐chain angles by Monte Carlo simulated annealing. Among the highest scored ROSETTA‐predicted models, we selected five models that agreed with NMR data …”

“…Although these NMR studies provided invaluable insight into the CB1 Ct peptide structure, it is necessary to determine the structure of CB1 Ct in contact with the G protein to gain structural information about the potential role of CB1 Ct in G protein signaling. In this study, we elucidated the plausible interactions of CB1 Ct with the G protein by building on our prior modeling effort of the CB1‐Gi complex lacking CB1 Ct (CB1( no Ct )‐Gi) .…”