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Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.pdf (1.9 MB)

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.

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posted on 2019-05-28, 10:30 authored by Frederike M. Müskens, Richard J. Ward, Dominik Herkt, Helmus van de Langemheen, Andrew B. Tobin, Rob M. J. Liskamp, Graeme Milligan
Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein-coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography-mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P-NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P-binding receptor via liquid chromatography-tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Funding

F.M.M. thanks the University of Glasgow for a Lord Kelvin-Adam Smith studentship.

History

Citation

Molecular Pharmacology, 2019, 95 (2), pp. 196-209

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

  • VoR (Version of Record)

Published in

Molecular Pharmacology

Publisher

American Society for Pharmacology and Experimental Therapeutics (ASPET)

eissn

1521-0111

Acceptance date

2018-11-27

Copyright date

2019

Available date

2019-05-28

Publisher version

http://molpharm.aspetjournals.org/content/95/2/196

Notes

This article has supplemental material available at molpharm.aspetjournals.org.

Language

en

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