@article{Wang:2024:10.1016/j.abb.2024.109944,
author = {Wang, X and Slater, A and Lee, SC and Harrison, N and Pollock, NL and Bakker, SE and Navarro, S and Nieswandt, B and Dafforn, TR and García, Á and Watson, SP and Tomlinson, MG},
doi = {10.1016/j.abb.2024.109944},
journal = {Arch Biochem Biophys},
title = {Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.},
url = {http://dx.doi.org/10.1016/j.abb.2024.109944},
volume = {754},
year = {2024}
}

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TY  - JOUR
AB  - The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.
AU  - Wang,X
AU  - Slater,A
AU  - Lee,SC
AU  - Harrison,N
AU  - Pollock,NL
AU  - Bakker,SE
AU  - Navarro,S
AU  - Nieswandt,B
AU  - Dafforn,TR
AU  - García,Á
AU  - Watson,SP
AU  - Tomlinson,MG
DO  - 10.1016/j.abb.2024.109944
PY  - 2024///
TI  - Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.
T2  - Arch Biochem Biophys
UR  - http://dx.doi.org/10.1016/j.abb.2024.109944
UR  - https://www.ncbi.nlm.nih.gov/pubmed/38395124
VL  - 754
ER  - 

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