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.
|Original language||English (US)|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Mar 16 2021|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS. This research was funded by the NIH (R35-GM126940 to S.S. and R01-GM117923 to N.V. and S.S.) and American Heart Association (18PRE34080117 to K.K.). We thank Daniel Hilger for sharing published data on GTP turnover.
© 2021 National Academy of Sciences. All rights reserved.
- FRET sensors
- G protein-coupled receptors
- Giant plasma membrane vesicles
- Ligand efficacy
- Molecular dynamics simulations