title: 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 (CB1(5P)(454-473)). Complexes formed between the peptide and human arrestin-2 (wt-arr2(1-418)) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2(1-382)) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1(5P)(454-473) adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh(7P)(330-348)), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1(5P)(454-473) peptide binds to tr-arr2(1-382) with higher affinity than to the full-length wt-arr2(1-418). 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 CB1(5P)(454-473) peptide.
authors.affiliation: Center for Drug Discovery, 360 Huntington Avenue, 116 Mugar Hall, Boston, Massachusetts 02115, United States.
authors.forename: Shubhadra N
authors.initials: SN
authors.lastname: Singh
authors.forename: Kunal
authors.initials: K
authors.lastname: Bakshi
authors.forename: Richard W
authors.initials: RW
authors.lastname: Mercier
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
authors.forename: Spiro
authors.initials: S
authors.lastname: Pavlopoulos
mesh_terms: D051046:Amino-Acid N-Acetyltransferase; D019390:Arrestins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; C000605257:Arrb2 protein, rat; D019390:Arrestins; C543264:Cnr1 protein, rat; D010446:Peptide Fragments; D043884:Receptor, Cannabinoid, CB1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D051046:Amino-Acid N-Acetyltransferase