Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors.

Rasenick, Mark M.; Watanabe, Masayuki; Lazarevic, Milenko B.; Hatta, Shinichi; Hamm, Heidi E.

doi:10.1016/s0021-9258(17)31835-5

Cited by 117 publications

(25 citation statements)

References 38 publications

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“…While binding affinity for the neutral antagonist I 125 -(±)-cyanopindolol is similar between GPMVs containing WT β2AR and β2AR-Spep, the latter shows significantly tighter binding to the full agonist Iso (∼40fold). The Gα C terminus is known to enhance agonist binding affinity (26). We have previously reported ∼50-fold enhanced Iso binding in crude membranes for β2AR-Spep compared to a β2AR SPASM sensor lacking the G-peptide (27).…”

Section: Resultsmentioning

confidence: 98%

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Kim

Paulekas

Sadler

et al. 2021

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

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Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

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Section: Resultsmentioning

confidence: 98%

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Kim

Paulekas

Sadler

et al. 2021

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

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“…Several tools including G-protein-specific pharmacological inhibitors or toxins [ 40 ], C-terminus mimicking peptides [ 41 ], small interfering RNA [ 42 , 43 ], using artificial systems with limited endogenous G-proteins [ 44 , 45 , 46 ] or reconstitution of purified receptors and G-proteins in the artificial membrane [ 47 , 48 ] limit the signal mediated by certain G-proteins. Techniques like the immunoprecipitation with specific Gα antibodies [ 2 , 49 ], resonance energy transfer techniques, where bioluminescent (BRET) or fluorescent (FRET) donors and acceptors are fused on the C-terminus of the GPCR and in one of the subunits of the G-protein [ 50 , 51 ] were used to study specific GPCR-G-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

Randáková

Nelic

Hochmalová

et al. 2021

IJMS

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A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

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“…[7a,20] To overcome the drawbacks encountered with the above protein-based technologies and thanks to the determination of receptor X-ray crystal structures,w ea nd others designed peptidomimetic ligands in an attempt to act as allosteric modulators targeting the G s protein binding site. [21] In an effort to investigate the docking event between the G s protein and b 2 AR, the groups of Hamm and Sejer-Pedersen used synthetic peptide analogues of the a subunit. [21b,c] It was demonstrated that some peptides were able to reduce cyclic adenosine monophosphate (cAMP) accumulation, which was indicative of binding to the receptor and consequently blocking the access of the Gp rotein.…”

Section: Introductionmentioning

confidence: 99%

Development of Generic G Protein Peptidomimetics Able to Stabilize Active State G_s Protein‐Coupled Receptors for Application in Drug Discovery

Mannes

Martin

Triest³

et al. 2021

Angewandte Chemie

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Gp rotein-coupled receptors (GPCRs) represent an important group of membrane proteins that play acentral role in modern medicine.U nfortunately,c onformational promiscuity hampers full therapeutic exploitation of GPCRs,since the largest population of the receptor will adopt ab asal conformation, which subsequently challenges screens for agonist drug discovery programs.H erein, we describe as et of peptidomimetics able to mimic the ability of Gp roteins in stabilizing the active state of the b 2 adrenergic receptor (b 2 AR) and the dopamine 1r eceptor (D1R). During fragment-based screening efforts,t hese (un)constrained peptide analogues of the a 5 helix in G s proteins,w ere able to identify agonism preimprinted fragments for the examined GPCRs,a nd as such, they behave as ag eneric tool, enabling an engagement in agonist earmarked discovery programs.

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Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors.

Cited by 117 publications

References 38 publications

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

Development of Generic G Protein Peptidomimetics Able to Stabilize Active State G_s Protein‐Coupled Receptors for Application in Drug Discovery

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Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors.

Cited by 117 publications

References 38 publications

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

Development of Generic G Protein Peptidomimetics Able to Stabilize Active State Gs Protein‐Coupled Receptors for Application in Drug Discovery

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Development of Generic G Protein Peptidomimetics Able to Stabilize Active State G_s Protein‐Coupled Receptors for Application in Drug Discovery