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Updated: Jul 31, 2025
07:41
A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
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Gαs slow conformational transition upon GTP binding and a novel Gαs regulator
Donghoon Ahn1, Davide Provasi2, Nguyen Minh Duc1
1School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Iscience
|May 2, 2023
Summary
G proteins mediate cell signaling. This study reveals rapid GTP binding and dissociation of Gαs from β2AR and Gβγ, followed by slow closing of the Gαs alpha-helical domain, with MAGE D2 accelerating this process.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- G proteins are key signaling molecules interacting with G protein-coupled receptors (GPCRs).
- Previous studies detailed GPCR-G protein complex formation and GDP release, but GTP binding dynamics remained unclear.
Purpose of the Study:
- To characterize the atomic-level conformational changes during GTP binding to the β2-adrenergic receptor (β2AR)-Gs complex.
- To develop a kinetic model for the β2AR-Gs complex post-GDP release and during GTP binding.
Main Methods:
- Bayesian integrative modeling framework.
- Hydrogen-deuterium exchange mass spectrometry (HDX-MS).
- Tryptophan-induced fluorescence quenching.
- Metadynamics simulations.
- Yeast-two-hybrid screening.
Main Results:
- GTP binding to the β2AR-Gs complex is rapid.
- GTP induces swift dissociation of Gαs from β2AR and Gβγ.
- The Gαs alpha-helical domain (AHD) closing is a slow process.
- Melanoma-associated antigen D2 (MAGE D2) was identified as a novel Gαs AHD-binding protein.
- MAGE D2 accelerates the GTP-induced closing of the Gαs AHD.
Conclusions:
- The study provides a detailed kinetic and structural model of GTP binding to the β2AR-Gs complex.
- MAGE D2 plays a regulatory role in G protein signaling by modulating Gαs AHD conformation.
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