TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome

Olsen, Reid H.J.; DiBerto, Jeffrey F.; English, Justin G.; Glaudin, Alexis M.; Krumm, Brian E.; Slocum, Samuel T.; Che, Tao; Gavin, Ariana C.; McCorvy, John D.; Roth, Bryan L.; Strachan, Ryan T.

doi:10.1038/s41589-020-0535-8

Cited by 391 publications

(508 citation statements)

References 44 publications

Supporting

Mentioning

496

Contrasting

Unclassified

Order By: Relevance

“…To this end we used an optimized protein proximity assay that detects receptor-stimulated release of Gβγ from Gα. In this bioluminescence resonance energy transfer (BRET) assay, a Gα- Renilla luciferase (RLuc) fusion is co-transfected with Gβ 3 and a Gγ 9 -GFP fusion (Olsen et al, 2020). For these experiments we used the neurotensin receptor NTR1 because it couples efficiently to multiple Gα subtypes including Gα i , Gα q and Gα s .…”

Section: Resultsmentioning

confidence: 99%

“…Bioluminescence Resonance Energy Transfer (BRET) assay. HEK293T cells (ATCC, #CRL-11268) were used to measure basal association and receptor-mediated dissociation of Gα and Gβγ, as described previously (Olsen et al, 2020). Briefly, cells were transfected with plasmids encoding the neurotensin or µ-opioid receptor, Gα fused to Renilla luciferase 8 (Rluc8), Gβ3 and γ9-fused to GFP in a 1:1:1:1 ratio (cell density 750,000 cells in 3 mL).…”

Section: Figure Legendsmentioning

confidence: 99%

See 1 more Smart Citation

A Universal Allosteric Mechanism for G Protein Activation

Knight

Ghosh

Campbell

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

SUMMARYG proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of Gα from the Gβγ subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This “G-R-E motif” secures GTP and, through an allosteric link, discharges the Gβγ dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation.HIGHLIGHTSReceptors promote GTP-GDP exchange and dissociation of G protein α and βγ subunitsWe find an allosteric network linking the γ phosphate of GTP with release of GβγThe network consists of a conserved Gly-Arg-Glu “activation triad”Triad mutations prevent subunit dissociation, regardless of agonist or GTP bindingTriad mutations are responsible for human endocrine and neurological disorders

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Figure Legendsmentioning

confidence: 99%

A Universal Allosteric Mechanism for G Protein Activation

Knight

Ghosh

Campbell

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The BRET recruitment assays were performed with HEK293T cells according to previously reported procedures with minor modifications [ 26 ]. Briefly, the cells were co-transfected overnight with Gα oA -RLuc, Gβ3, Gγ8-GFP2 and human KOR receptor at a 1:1:1:1 ratio.…”

Section: Methodsmentioning

confidence: 99%

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor

Nieman

Mian

et al. 2020

Molecules

View full text Add to dashboard Cite

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC50 of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with 3H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAAR) of the α1-3β2-3γ1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABAARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.

show abstract

“…According to the activation parameters, an MSM was built using the PyEMMA protocol (2) Gq protein activation Gq BRET probes were generated according to a previous report 93 . HEK293T cells were transiently co-transfected with AT1R and Gq BRET probes.…”

Section: Markov State Model Constructionmentioning

confidence: 99%

Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as novel targets for allosteric drug design

Yang

Chai

et al. 2020

Preprint

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) are the most frequent targets of approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we utilized a combined computational and experimental framework that integrated extensive molecular dynamics simulations, Markov state models, and site-directed mutagenesis for investigating the conformational landscape of activation of the angiotensin II (AngII) type 1 receptor (AT1R), a prototypical class A GPCR. Our findings suggested a synergistic transition mechanism of AT1R activation. Importantly, a key intermediate state was found in the activation pathway. We discovered a novel “cryptic” binding site in the intracellular region of the receptor in the intermediate state. Furthermore, a bioluminescence resonance energy transfer analysis revealed the insensitivity of the endogenous AngII octapeptide agonist in the activation of the downstream Gq signaling and β-arrestin-mediated pathways, upon mutations of the predicted cryptic binding site, thereby suggesting an allosteric regulatory mechanism. Together, these findings not only provide a deeper understanding of AT1R activation at an atomic level, but also open potential avenues for the design of allosteric AT1R modulators. Consequently, this will provide a broad range of applications for GPCR biology, biophysics, and medicinal chemistry.

show abstract

TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome

Cited by 391 publications

References 44 publications

A Universal Allosteric Mechanism for G Protein Activation

A Universal Allosteric Mechanism for G Protein Activation

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor

Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as novel targets for allosteric drug design

Contact Info

Product

Resources

About