Thermodynamic role of receptor phosphorylation barcode in cannabinoid receptor desensitization

Abstract

The endocannabinoid signaling system is comprised of CB1 and CB2 G protein-coupled receptors (GPCRs). CB2 receptor subtype is predominantly expressed in the immune cells and signals through its transducer proteins (Gi protein and β-arrestin-2). Arrestins are signaling proteins that bind to many GPCRs after receptor phosphorylation to terminate G protein signaling (desensitization) and to initiate specific G protein-independent arrestin-mediated signaling pathways via a "phosphorylation barcode", that captures sequence patterns of phosphorylated Ser/Thr residues in the receptor's intracellular domains and can lead to different signaling effects. The structural basis for how arrestins and G proteins compete with the receptor for biased signaling and how different barcodes lead to different signaling profiles is not well understood as there is a lack of phosphorylated receptor structures in complex with arrestins. In this work, structural models of β-arrestin-2 were built in complex with the phosphorylated and unphosphorylated forms of the CB2 receptor. The complex structures were relaxed in the lipid bilayer environment with molecular dynamics (MD) simulations and analyzed structurally and thermodynamically. The β-arrestin-2 complex with the phosphorylated receptor was more stable than the non-phosphorylated one, highlighting the thermodynamic role of the receptor phosphorylation. It was also more stable than any of the G protein complexes with CB2 suggesting that phosphorylation signals receptor desensitization (end of G protein signaling) and arrest of the receptor by arrestins. These models are beginning to provide the thermodynamic landscape of CB2 signaling, which can help bias signaling towards therapeutically beneficial pathways in drug discovery applications.

Keywords: Endocannabinoid system; GPCRs; Molecular dynamics; Phosphopeptide; Signaling complexes.

Fig. 1.

A. GPCR mediated G-protein-dependent signaling and G-protein-independent (β-arrestin) signaling complexes; B. Structure of CB2:Gi-GDP protein complex based on PDB 6KPF; C. PDB structure 6TKO of β-arrestin-1 (Blue) interacting with a phosphorylated peptide (Yellow).

Fig. 2.

A. The structure building and simulation protocol; B. Alignment of available β-arrestin-1 structures in complex with receptors; PDB: 7R0C (cyan), 6TKO (green/yellow), 6U1N (pink), 7SRS (salmon).

Fig. 3.

A. CB2-NoPhosphoC:β-arrestin-2; B. CB2-PhosphoC:β-arrestin-2; C. CB2-NoPhosphoC:Gi-GDP; D. CB2-NoPhosphoC:Gi-Empty; E. MMPBSA-based free-energy of binding of transducer (G protein/β-arrestin-2) for CB2.

Fig. 4.

Average snapshots of the A. CB2-NoPhosphoC:Gi-GDP complex; B. CB2-NoPhosphoC:β-arrestin-2 complex; C. PhosphoC site interface of the CB2-PhosphoC:β-arrestin-2 complex; D. G protein coupling site interface of the CB2-PhosphoC:β-arrestin-2 complex. E. CB2:Transducer Binding Free-Energy Landscape of CB2 signaling. The connections between states and barriers are qualitative, hence shown with dashes.