A Key Agonist-induced Conformational Change in the Cannabinoid Receptor CB1 Is Blocked by the Allosteric Ligand Org 27569

Fay, Jonathan F.; Farrens, David

doi:10.1074/jbc.m112.352328

Cited by 44 publications

(72 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Previously, using SDFL, we discovered that Org 27569 blocks TM6 movement (10). However, in that work, we could not unequivocally determine if the Org 27569 bound structure was the same or different from the antagonist bound form.…”

Section: Org 27569 Favors a Receptor With An Inactive Conformation Andmentioning

confidence: 85%

“…In contrast, the allosteric ligand enhances the fluorescence changes for the probe at H8/TM7, whereas it blocks agonist-induced changes at TM6 (Fig. 4) (10).…”

Section: Sdfl Studies Confirm That Org 27569 Traps the Receptor In A mentioning

confidence: 95%

“…An outward movement of TM6 is critical for G-protein activation, because it exposes a hydrophobic binding site and enables formation of the ternary complex of receptor, ligand, and G protein (4)(5)(6)(7)(8)(9). Newer evidence suggests that there is likely some plasticity in TM6 movement during activation, with differences in either the magnitude or probability of the movement explaining varying degrees of G-protein signaling (3,10,11).…”

mentioning

confidence: 99%

“…It also enables well-characterized radioactively labeled orthosteric CB 1 ligands to be used. Second, Org 27569 exhibits a number of unusual effects-it increases agonist binding to the receptor while simultaneously inhibiting G-protein activation (10,22), and inducing biased signaling (23)(24)(25). Thus, it is hard to imagine how these different effects could occur unless the CB 1 -Org 27569 bound state adopts a unique and different conformation.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Fay

Farrens

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Org 27569 Favors a Receptor With An Inactive Conformation Andmentioning

confidence: 85%

“…In contrast, the allosteric ligand enhances the fluorescence changes for the probe at H8/TM7, whereas it blocks agonist-induced changes at TM6 (Fig. 4) (10).…”

Section: Sdfl Studies Confirm That Org 27569 Traps the Receptor In A mentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Fay

Farrens

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Labeled ATR exchange was done by photoactivating regenerated opsins in the presence of exogenous [ 3 H]ATR. Unbound radiolabeled retinals were removed using mini size-exclusion chromatography columns and the incorporated [ 3 H]ATR was counted by liquid scintillation (43). See SI Experimental Procedures for full details.…”

Section: Methodsmentioning

confidence: 99%

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

Schafer

Fay

Janz

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on timeresolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.T he superfamily of G protein-coupled receptors (GPCRs) is one of the largest targets of pharmaceutical drugs in the human genome. Classically, GPCR signaling occurs when a diffusible ligand (such as a drug) binds to the receptor and stabilizes conformations that can couple with and activate intracellular proteins. Our understanding of this process has built on the classical "ternary complex" model of receptor-ligand-G protein interaction (1), a model that, with revisions, has continued to guide our knowledge of how this critical event occurs.However, this paradigm has faced problems when applied to rhodopsin, the dim-light visual receptor. Rhodopsin is kept in an "off" state by a covalently bound inverse agonist, 11-cis retinal (11CR). Light converts the 11CR to an agonist, all-trans retinal (ATR), which enables the receptor to activate its G protein, transducin (G t ) (2, 3). The active receptor, metarhodopsin II (MII), continues signaling until the Schiff base linking ATR to the receptor is hydrolyzed, resulting in the release of ATR and the decay of MII into an inactive apoprotein, opsin (4, 5). Binding of a new 11CR to opsin reforms the dark state (DS), enabling another round of photon detection (6).Due to this unusual light-activated, covalently bound ligand, rhodopsin has usually been considered "different" from the larger superfamily of diffusible ligand-binding GPCRs. However, we recently discovered that rhodopsin behaves more like a traditional ligand-binding GPCR than previously thought (7). Our expe...

show abstract

In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

et al. 2015

View full text Add to dashboard Cite

We present in-membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G-protein-coupled receptors (GPCRs) with minimal mutagenesis.T his method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site-specific introduction of chromophores for NMR and fluorescence spectroscopyinthe human k-opioid receptor (KOR) and the human A 2A adenosine receptor (A 2A AR). IMCM is applicable to aw ide range of in vitro studies of GPCRs,including single-molecule spectroscopy, and is apromising platform for in-cell spectroscopyexperiments.

show abstract

A Key Agonist-induced Conformational Change in the Cannabinoid Receptor CB1 Is Blocked by the Allosteric Ligand Org 27569

Cited by 44 publications

References 60 publications

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

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

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

In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

Contact Info

Product

Resources

About

A Key Agonist-induced Conformational Change in the Cannabinoid Receptor CB1 Is Blocked by the Allosteric Ligand Org 27569

Cited by 44 publications

References 60 publications

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

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

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

In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

Contact Info

Product

Resources

About

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

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