Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

Abstract

The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoylsn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

The A2aR C‐terminus provides improved total and active expression yields for adenosine receptor chimeras

“…Whole cell ligand binding was performed with receptor‐specific antagonist. For A 2a R, ligand binding with agonist as compared to antagonist showed lower B max values (data not shown), which is consistent with previous reports . Therefore, all further experiments were performed using antagonist to determine the total surface‐localized receptor capable of ligand binding.…”

A2AR Binding Kinetics in the Ligand Depletion Regime

“…as described previously 16, 30 ). Because of the low experimental variability in equilibrium K D , and the close fit between model and experimental data, the fixed K D approach was used in the remaining one-ligand binding numerical analysis, and is recommended for those implementing this technique.…”

Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus

“…The yeast S. cerevisiae is a microbial eukaryotic host uniquely positioned to produce functional GPCRs and to characterize downstream signaling. For example, functional GPCRs have been purified from S. cerevisiae [ 12 , 13 , 14 ] and human GPCRs can signal upon ligand binding via an engineered MAPK response pathway [ 15 , 16 , 17 ] enabling identification of novel drug candidates [ 18 ]. In addition, yeast have been utilized to study GPCR-Gα protein interactions [ 19 , 20 ] and the effect of receptor dimerization on signaling responses [ 21 ].…”