Evolutionary action and structural basis of the allosteric switch controlling β₂AR functional selectivity.

Area of Science:

• Pharmacology
• Molecular Biology
• Biophysics

Background:

• G-protein-coupled receptors (GPCRs) exhibit functional selectivity, activating specific signaling pathways based on ligand-induced conformations.
• Understanding the molecular basis of this selectivity is crucial for drug development.

Purpose of the Study:

• To identify molecular motifs responsible for transducing ligand binding into distinct signaling responses in GPCRs.
• To elucidate the structural underpinnings of functional selectivity in the beta-2 adrenergic receptor.

Main Methods:

• In silico evolutionary lineage analysis.
• Structure-guided site-directed mutagenesis.
• Large-scale functional signaling characterization.
• Non-negative matrix factorization clustering of signaling profiles.

Main Results:

• Clustering of 28 beta-2 adrenergic receptor variants revealed three distinct phenotypic clusters.
• These clusters showed selective impairments in Gi or beta-arrestin/endocytosis pathways, with preserved Gs activation.
• Mutations affecting DRY, NPxxY, and PIF motifs were linked to specific signaling profiles.

Conclusions:

• Specific receptor regions and micro-switches (DRY, NPxxY, PIF motifs) are critical for stabilizing distinct conformations.
• These distinct conformations underlie the observed functional selectivity of GPCRs.
• The study provides insights into the molecular mechanisms governing biased agonism in GPCR signaling.