Biology subjects

Wingler, L. M.

Publications and source records attributed to Wingler, L. M..

4 recordsLinked to original sources

bioRxiv2026

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

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 G subunits that diverge only in the C-terminal 5 helix, we identify four residues dispersed throughout the 5 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 5 helix increase affinity for AngII-activated AT1R. We provide evidence that the 5 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.

biochemistry↗

bioRxiv2025

Angiotensin receptor conformations stabilized by biased ligands differentially modulate β-arrestin interactions

"Biased" ligands of the angiotensin II type 1 receptor (AT1R) preferentially activate G protein or {beta}-arrestin pathways by stabilizing distinct receptor conformations. Here we show that {beta}-arrestin-biased AT1R ligands further vary in their ability to stabilize different modes of {beta}-arrestin interaction, specifically interactions with the AT1R seven-transmembrane core versus the phosphorylated C-terminus. By combining biochemical assays with double electron-electron resonance (DEER) spectroscopy and integrative modeling, we show that ligands less effective at stabilizing the core complex promote an AT1R conformation with an intermediate transmembrane helix 6 position that is incompatible with {beta}-arrestin core binding. Since the core and phosphosite interactions differentially activate the signaling, internalization, and desensitization functions of {beta}-arrestin, our data demonstrate that the allosteric effects of GPCR ligands could directly modulate {beta}-arrestin activities. This "intra-transducer bias," or bias toward various functions of the same transducer, could enable finer control of GPCR drugs pharmacology than previously thought possible.

biochemistry↗

bioRxiv2023

Antibodies Expand the Scope of Angiotensin Receptor Pharmacology

G protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue, and cellular level. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays, and structural studies, we develop maternally selective heavy chain-only antibody ("nanobody") antagonists against the angiotensin II type I receptor (AT1R) and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to AT1R with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

pharmacology and toxicology↗

bioRxiv2020

Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naive nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw [~]580-fold and [~]925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

bioengineering↗