An Affinity-Based Probe for the Human Adenosine A_2A Receptor.

Area of Science:

• Biochemistry
• Chemical Biology
• Pharmacology

Background:

• Activity-based protein profiling (ABPP) allows functional protein interrogation in native environments.
• G protein-coupled receptors (GPCRs) are pharmaceutically important but challenging targets for ABPP.
• The human adenosine A2A receptor (hA2AR) serves as a model GPCR for developing new ABPP tools.

Purpose of the Study:

• To construct a chemical toolbox for two-step affinity-based labeling of GPCRs.
• To develop a novel probe for the human adenosine A2A receptor (hA2AR) using ABPP.
• To establish a method for visualizing and quantifying GPCRs in biological samples.

Main Methods:

• Synthesis of an irreversibly binding hA2AR ligand functionalized with a terminal alkyne for probe development.
• Affinity-based labeling of purified hA2AR and hA2AR in cell membranes.
• Click-ligation with a sulfonated cyanine-3 fluorophore for visualization.
• Inhibition studies using selective antagonists to confirm probe specificity.

Main Results:

• The developed probe demonstrated irreversible and concentration-dependent labeling of purified hA2AR.
• Successful visualization of labeled hA2AR on SDS-PAGE via click chemistry.
• Labeling of hA2AR was inhibited by selective antagonists, confirming probe specificity.
• The probe effectively labeled hA2AR in cell membranes overexpressing the receptor.

Conclusions:

• The novel chemical probe is a promising tool for affinity-based labeling of GPCRs.
• This work lays the foundation for developing advanced ABPP probes for various GPCRs.
• The methodology enables functional interrogation of GPCRs in complex biological systems.