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Solubilization of stable adenosine A1 receptors from rat brain.

ABSTRACT: Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.

SUBMITTER: Helmke SM

PROVIDER: S-EPMC1135595 | biostudies-other | 1989 Jan

REPOSITORIES: biostudies-other

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Project description:Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

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Solubilization of stable adenosine A1 receptors from rat brain.

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