Structural basis of human cannabinoid CB1 G protein‐coupled receptor activation

Abstract

Transmembrane signal transduction is achieved by activation of G protein‐coupled receptors (GPCRs) like the human cannabinoid CB1 receptor. These receptors exist in an ensemble of conformations, each of which might bind to different signaling molecules. Mutating a single residue, Thr 210, to Ile in the third transmembrane (TM3) domain, makes it more active then WT, whereas mutating it to Ala makes it fully inactive. We used the Gensemble method to predict 3D structures of these receptors. We find conformational differences that explain the CB1 receptor's activation mechanism. These predictions were validated by designing double mutants that were expected to switch the inactive T210A back to WT levels of activation. The 2nd mutation for T210A is predicted to cause an important salt‐bridge between TMs 2 and 6 to break. GTPδS binding assays show a large increase in G protein‐coupling for the double mutants indicating increased activation. We docked known agonists to these receptors and then performed 50 ns of molecular dynamics. The inactive T210A receptor with the docked agonist WIN55212‐2 maintains two stable interhelical salt‐bridges. Similarly, the WT receptor maintains a salt‐bridge between TMs 3 and 6, which suggests that a G protein is necessary to stabilize the active conformation.This work was funded by gifts from PharmSelex/Accelerator and NIH grants (R01NS071112, R01NS073115, R01AI040567, and DA020763).