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Purification of the Active-State G Protein-Coupled Receptor ADGRL4 for Cryo-Electron Microscopy Using a Modular Tag
David M Favara1,2,3, Christopher G Tate1
1Division of Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
Bio-Protocol
|March 12, 2026
Summary
We developed a rapid workflow to purify active adhesion G protein-coupled receptor 4 (ADGRL4) for structural studies. This method enhances receptor stability and expression, aiding research into cancer progression.
Area of Science:
- Structural biology
- Biochemistry
- Molecular biology
Background:
- Adhesion G protein-coupled receptor 4 (ADGRL4) is involved in tumor progression.
- Previous studies determined the active-state cryo-EM structure of ADGRL4, showing weak coupling with Gq protein.
Purpose of the Study:
- To establish a modular workflow for purifying active-state ADGRL4.
- To enhance receptor stability and cell-surface expression for structural analysis.
Main Methods:
- Utilized a multifunctional N-terminal tagging strategy with orthogonal purification and detection tags.
- Incorporated a tethered mini-Gq at the C-terminus to stabilize the receptor.
- Developed a rapid (2-3 day) purification protocol.
Main Results:
- Achieved enhanced cell-surface expression and stability of ADGRL4.
- Demonstrated the modularity of the tagging strategy, allowing flexibility in purification methods.
- Successfully purified active-state ADGRL4 for cryo-EM analysis.
Conclusions:
- The described workflow provides an efficient method for purifying active-state ADGRL4.
- This approach is transferable to other challenging G protein-coupled receptors (GPCRs) requiring enhanced stability for structural studies.
