Exploring structural mechanism for the regulation of CB1 desensitization and internalization

Arrestins are cytosolic proteins that specifically bind to G-protein coupled receptors (GPCRs), thereby inhibiting the GPCR-G-protein signaling interactions. Interactions between arrestins and Cannabinoid receptor 1 (CB1, a member of the GPCR superfamily), that lead to CB1 desensitization and internalization are of significant clinical interest, particularly due to their role in development of tolerance. Although arrestin-CB1 interactions have been examined by mutational studies, structural information on these complexes is lacking. The work reported in this thesis focuses on structural elucidation of arrestin-2-CB1 interactions. ^ Based on existing data, the role of CB1 C-terminus phosphorylation on the nature of the CB1-arrestin interactions, and the functional consequences of desensitization and internalization of CB1 are unclear. To gain structural insight into this system we have used differentially phosphorylated peptide mimics of CB1 cytosolic segments and employed NMR methods to study the structural details of their interaction with arrestin-2. The investigation of these interactions is complicated by the fact that a conformational change is also expected in arrestin-2 during these interactions. Initially, displacement of the C-terminus tail, away from the arrestin-2 molecule occurs, preceding potentially more significant arrestin conformational changes. Hence, we have also used the arrestin-2 C-tail truncated mutant for better understanding of CB1-arrestin interactions. ^ Structural studies of CB1 peptides suggest that CB1 segments implicated in arrestin interaction are flexible in solution and during our investigation, we found that these segments undergo a significant conformational change to achieve the functionally bioactive conformation. This conformation is a result of both, phosphorylation as well as the interaction with arrestin. We found that increase in CB1 phosphorylation expectedly led to a higher affinity complex between arrestin-2 and CB1.^ Our studies of the CB1–arrestin desensitization complex found that arrestin binding induced helices in the vicinity of the two-phosphorylation sites. Truncation of arrestin C-tail led to loss of this CB1 peptide-complex indicating that the arrestin basal state must be maintained for this interaction. ^ The studies of CB1-arrestin internalization complex found that a turn inherent to the primary amino acid sequence, localized over the phosphorylation motif on the CB1 distal tail, plays a primary role in this CB1-arrestin interaction. Furthermore, we found that the removal of the arrestin C-tail led to a higher affinity complex between CB1 peptide and arrestin. This indicates that a significant conformational change in arrestin conformation is required for internalization, and the phosphorylation-sensitive residues on the arrestin N-domain participate in binding. The idea of conformational change was supported by thermal denaturation studies that demonstrated a change in arrestin-2 (1-382) heat capacity upon binding with CB1 internalization peptide. ^ These studies thus provide novel structural information on arrestin-GPCR interactions and the role of receptor phosphates in these interactions. This work also provides a model for subsequent studies on arrestin-GPCR interactions.