Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB ₁

Abstract

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

“…Conformational selectionbased ligand-binding mechanisms as well as multiple receptor signaling states are well documented for other GPCRs, including the B2AR and CB1 receptor (7,20,(35)(36)(37)(38). Hence, the delayed reversion to the inactive receptor conformation we observe for rhodopsin might play a heretofore unappreciated role here and in other GPCR signaling systems.…”

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

“…Only G-protein binding results in the complete transition to the active conformation, which essentially abolishes the aforementioned dynamics18. This and other data suggests that the assumption of just one active receptor structure may represent a too simplistic representation of the function of GPCRs9.…”

“…The most important dynamic structural alteration has been observed for helix 6 of the heptahelical membrane proteins. Specific labeling of the protein with fluorescence910, EPR1112, or NMR13141516171819 probes have highlighted the dynamical alterations of the molecule upon inverse agonist or agonist binding as well as G-protein interaction. The most complete set of data that describes the interesting structural dynamics of GPCRs is available for the β 2 -adrenergic receptor (β 2 AR).…”

Modulation of CB1 cannabinoid receptor by allosteric ligands: Pharmacology and therapeutic opportunities

“…As one way to address this question, Farrens and colleagues utilized site directed fluorescence labeling techniques to investigate changes in transmembrane helix (TM) movements upon binding of ORG27569 (Fay et al, 2015). They discovered that upon binding to the CB 1 receptor, the outward TM6 movements that are necessary to activate the receptor by exposing the binding site crevice for G-protein are blocked by ORG27569, thus explaining why ORG27569 inhibits G-protein coupling (Fay et al, 2012).…”