A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

Tsukamoto, Hisao; Farrens, David

doi:10.1074/jbc.m113.472464

Cited by 41 publications

(62 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the effect of these mutations could be because of altering one or more steps on this activation pathway and not simply the breaking or making of one hydrogen bond. Interestingly, although not directly comparable, these energy differences are of a similar magnitude as the activation energy barrier for TM6 movement previously measured for the corresponding CAM mutation in rhodopsin (38). Another important caveat regarding our pharmacological studies-although they provide a good first approximation for assessing the receptor behavior with regards to agonist binding and G-protein activation, our modified allosteric ternary complex model cannot discern if more than one type of G protein-inactive state (such as R, R′, and R″) is present.…”

Section: Discussionmentioning

confidence: 70%

See 1 more Smart Citation

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: Discussionmentioning

confidence: 70%

“…The mutant-θ is a nonreactive, minimal cysteine construct that enables fluorescence labeling for SDFL studies and has N-and C-terminal deletions to facilitate purification (10). Transfection was carried out transiently in COS-1 cells (10,38). Mutants used for SDFL studies were grown in the presence of 100 nM SR141716A.…”

Section: Methodsmentioning

confidence: 99%

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

“…In contrast, rhodopsin samples that contain active Ops* due to a constitutively activating mutation M257Y (CAM) (14,15) do not show full ATR release. Instead, ∼60% of the CAMs have ATR still bound under steady-state conditions (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…Opsin experiments contained a stabilizing disulfide (N2C/D282C) to facilitate apoprotein purification (42). Proteins were expressed by transient transfection in COS-1 cells and either purified as opsin or regenerated with 11CR and labeled with the fluorophore monobromobimane as previously described (8,15). 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

“…Protein Expression and Purification-The melanopsin mutants and jumping spider Rh1 were transiently expressed in COS-1 cells (typically 10 -20 plates), and the cells were harvested 48 h after transfection as described previously (34). The collected cells were incubated with 11-cis-retinal overnight, and membrane proteins were solubilized with 1.25% dodecyl maltoside (DDM; Dojindo, Kumamoto, Japan), 20 mM HEPES, 140 mM NaCl, 0.25% cholesterol hemisuccinate (Sigma) 25 mM Tris, 10% glycerol, pH 7.0.…”

Section: Construction and Expression Of Mutants Of Melanopsins Andmentioning

confidence: 99%

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Tsukamoto

Kubo

Farrens

et al. 2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Melanopsin is involved in non-visual photoreception in mammals, but molecular characteristics underlying these non-visual functions are unclear. Results: Stability of the bond with the retinal chromophore is weakened and diversified in mammalian melanopsins. Conclusion: Mammalian melanopsins have acquired characteristics suited for their non-visual functions. Significance: The weaker retinal attachment in melanopsin may contribute to the functional tuning of non-visual photoreception in mammals.

show abstract

A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

Cited by 41 publications

References 39 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

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Contact Info

Product

Resources

About

A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

Cited by 41 publications

References 39 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

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

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