{"doi":"10.1073/pnas.2017201118","title":"β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus","abstract":"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.","journal":"Proceedings of the National Academy of Sciences","year":2021,"id":175222,"datarank":0.47032413238937254,"base_score":3.1354942159291497,"endowment":3.1354942159291497,"self_citation_contribution":0.47032413238937254,"citation_network_contribution":0.0,"self_endowment_contribution":0.47032413238937254,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":22,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9537,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":716302,"name":"Shayla Paulekas","orcid":null,"position":1,"is_corresponding":false},{"id":715699,"name":"Fredrik Sadler","orcid":"0000-0002-3627-1940","position":2,"is_corresponding":false},{"id":428125,"name":"Tejas M. Gupte","orcid":"0000-0001-7422-016X","position":3,"is_corresponding":false},{"id":425586,"name":"Michael Ritt","orcid":null,"position":4,"is_corresponding":false},{"id":669042,"name":"Matthew Dysthe","orcid":"0000-0002-6559-3498","position":5,"is_corresponding":false},{"id":272488,"name":"Nagarajan Vaidehi","orcid":"0000-0001-8100-8132","position":6,"is_corresponding":false},{"id":424957,"name":"Sivaraj Sivaramakrishnan","orcid":"0000-0002-9541-6994","position":7,"is_corresponding":false},{"id":716301,"name":"Keehun Kim","orcid":null,"position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:47:11.249697Z","pmid":"33836582","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}