TY - JOUR
T1 - β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus
AU - Kim, Keehun
AU - Paulekas, Shayla
AU - Sadler, Fredrik
AU - Gupte, Tejas M.
AU - Ritt, Michael
AU - Dysthe, Matthew
AU - Vaidehi, Nagarajan
AU - Sivaramakrishnan, Sivaraj
N1 - Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/3/16
Y1 - 2021/3/16
N2 - 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.
AB - 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.
KW - FRET sensors
KW - G protein-coupled receptors
KW - Giant plasma membrane vesicles
KW - Ligand efficacy
KW - Molecular dynamics simulations
UR - http://www.scopus.com/inward/record.url?scp=85102340549&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85102340549&partnerID=8YFLogxK
U2 - 10.1073/pnas.2017201118
DO - 10.1073/pnas.2017201118
M3 - Article
C2 - 33836582
AN - SCOPUS:85102340549
SN - 0027-8424
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 11
M1 - e2017201118
ER -