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Purification of the Active-State G Protein-Coupled Receptor ADGRL4 for Cryo-Electron Microscopy Using a Modular Tag System and a Tethered mini-Gq
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DOI：10.21769/BioProtoc.5617.png)
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Abstract
En 中文
ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in tumour progression in multiple malignancies. We recently determined the first cryo-EM structure of active-state ADGRL4, revealing its weak coupling to the heterotrimeric G protein Gq and providing insights into its activation mechanism. Here, we describe a complete modular workflow for purifying active-state ADGRL4 over 2-3 days using a multifunctional tagging strategy incorporating multiple orthogonal detection, purification, and cleavage tags at the N-terminus as well as a tethered mini-Gq at the C-terminus. This configuration enhanced receptor cell-surface expression and stability and allowed different purification strategies to be tested during the development of the purification protocol. Although developed and optimised for ADGRL4, this approach is readily transferable to other weakly coupling aGPCRs or GPCRs where complex stability is a limiting factor for structural analysis.
Keywords:
Adhesion GPCR
aGPCR
ADGRL4
ELTD1
GPCR purification
G protein-coupled receptor
cryo-EM
Journal
B
IF:
1.1
Papers:
191
Citations:
5.4K
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Cited Papers