Agonist and antagonist interactions with the different functional states of the human cannabinoid CB1 receptor

Abstract

There is increasing experimental and theoretical evidence that G protein-coupled receptors (GPCRs) exhibit an ensemble of conformations, each of which could bind to different ligands and lead to different responses. At thermal equilibrium, the inactive conformation is energetically favorable, but binding to an agonist can stabilize the G protein-coupled state. For the cannabinoid CB1 receptor, mutating a threonine residue (T210) on the third transmembrane helix to an alanine changes the CB1 receptor to a fully inactive conformation that does not activate the G protein. However, the T210I mutation changes it to an activated conformation with dramatically increased constitutive activity (1). To understand these experimental observations, we used the Gensemble Monte Carlo method to predict the ensemble of low-lying conformations of these three receptors. To validate these predicted conformations, we docked known ligands to them using the DarwinDock hierarchical docking method. Our results explain the site-directed mutagenesis data. We find that the inverse agonist rimonabant is anchored by two hydrogen bonds to W279, which explains the experimental importance of this residue. The agonist WIN55212-2 forms an aromatic stack with this W279 and F200, both of which have been shown experimentally to be important. We further investigated how the T210A or the WT conformations would change in the presence of an agonist with 20 ns of molecular dynamics (NAMD) using a fully solvated membrane. Neither of these receptors changed to resemble the active T210I mutant. The WT receptor formed the signature ionic locks found in the inactive T210A receptor. The computational docking results of agonist WIN55212-2 suggest that it prefers to bind to the receptor's inactive conformation. This is consistent with the recently crystallized beta 1 and beta 2 adrenergic receptors in the absence of G proteins with a bound agonist. This work was initiated with gifts from PharmSelex/Accelerator and finished with funding from NIH grants (R01NS071112, R01NS073115, and R01AI040567). 1. D'Antona A, Ahn KH, & Kendall DA (2006) Mutations of CB1 T210 produce active and inactive receptor forms: Correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry 45(17):5606-5617.

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