Illuminating G-Protein-Coupling Selectivity of GPCRs

Area of Science:

• Molecular Biology
• Cellular Signaling
• Biochemistry

Background:

• Heterotrimeric G proteins (Gα, Gβ, Gγ) are key signal transducers, with four subfamilies (G_s, G_i/o, G_q/11, G_12/13).
• G-protein-coupled receptors (GPCRs) initiate signaling cascades by interacting with specific Gα C termini.
• Understanding the sequence determinants of GPCR-G protein coupling selectivity is crucial for deciphering cellular responses.

Purpose of the Study:

• To systematically quantify ligand-induced interactions between a comprehensive set of GPCRs and all Gα subunit C termini.
• To identify sequence-based features that dictate GPCR-G protein coupling specificity.
• To develop a predictive model for GPCR-G protein coupling and engineer selective GPCRs.

Main Methods:

• Utilized a transforming growth factor-α (TGF-α) shedding assay in HEK293 cells to probe chimeric Gα subunit activation.
• Complemented coupling profiles using a NanoBiT-G-protein dissociation assay.
• Developed and validated a computational predictor based on identified sequence features.

Main Results:

• Generated a large-scale dataset of GPCR-Gα subunit interactions for 148 GPCRs and 11 Gα C termini.
• Identified sequence-based coupling specificity determinants located both within and outside the transmembrane domain.
• Developed a novel coupling predictor that demonstrates superior performance compared to existing methods.
• Successfully engineered designer GPCRs with selective coupling to the G₁₂ subfamily.

Conclusions:

• The study provides a valuable, high-resolution dataset of GPCR signaling mechanisms.
• Sequence features outside the transmembrane domain significantly influence GPCR-G protein coupling specificity.
• The developed predictor and engineered GPCRs offer powerful tools for future research in GPCR signaling and drug discovery.