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

Keehun Kim, Shayla Paulekas, Fredrik Sadler, Tejas M. Gupte, Michael Ritt, Matthew Dysthe, Nagarajan Vaidehi, Sivaraj Sivaramakrishnan
2021 Proceedings of the National Academy of Sciences of the United States of America  
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
more » ... f 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.
doi:10.1073/pnas.2017201118 pmid:33836582 fatcat:7mr5dit4gjcb3eeep6q6sjmrii