Molecular Determinants of G Protein Subtype-Specific Coupling to the Angiotensin II Type 1 Receptor.

UNLABELLED: Many G protein-coupled receptors (GPCRs) are capable of activating multiple heterotrimeric G protein subtypes. The angiotensin II type 1 receptor (AT1R) couples to both Gq and Gi family members, and the two families mediate distinct aspects of the effects of angiotensin II (AngII) on blood pressure and fluid balance. Previous work has pointed to fundamental differences in the strength and ligand-dependence of Gq versus Gi activation by AT1R. Here we resolve key aspects of the mechanisms underlying these subtype-specific patterns of G protein coupling to AT1R. Using cellular biosensors, we show that AT1R activates Gq more efficaciously than Gi. Leveraging mini-G proteins, engineered versions of Gα subunits that diverge only in the C-terminal α5 helix, we identify four residues dispersed throughout the α5 helix that are sufficient to account for these differences. We employ radioligand binding to demonstrate that subtype-specific differences originate at the level of allosteric coupling between ligand and transducer binding, and we use Systematic Protein Affinity Strength Modulation biosensors to show that Gq-specific residues in the α5 helix increase affinity for AngII-activated AT1R. We provide evidence that the α5 helix residue with the greatest effect on coupling makes interactions with AT1R intracellular loop 3 that were not resolved in a previously reported AT1R-Gq cryo-electron microscopy structure. These findings enhance our understanding of how AT1R differentially activates Gq and Gi and may ultimately guide pharmacological approaches to modulate certain G proteins downstream of AT1R and other GPCRs.

SIGNIFICANCE STATEMENT: This study identifies four residues within the Gα C-terminal α5 helix that govern subtype-specific patterns of Gq and Gi coupling to the angiotensin II type 1 receptor. Our findings demonstrate that allosteric coupling dictates the differential activation patterns of these two G heterotrimeric proteins, advancing our understanding of transducer selectivity and guiding the development of targeted subtype-selective therapeutics.