Guanosine-5'-triphosphate (GTP)-binding protein-coupled receptors are the target of up to 40% of prescribed medications worldwide. To evaluate the suitability of novel receptor ligands, frequently elaborate, time-consuming, and expensive receptor-ligand interaction studies have to be carried out. This work describes the development and proof of principle of a rapid, sensitive, and reliable receptor-ligand binding assay. CHO cells were stably transfected with a construct encoding the human A1 adenosine receptor (hA1AR). For ligand binding assays, membranes from these cells were prepared and embedded in low melting point agarose. These "immobilized" samples were incubated with tritiated 8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX), a well-established receptor antagonist. The KD and Bmax values as well as kinetic parameters (kon and koff) of receptor-ligand interaction were determined. Unspecific binding of various radiotracers to either the carrier material or the agarose gel matrix was negligible. The dissociation constant (KD) for [3H]DPCPX at the hA1AR was determined by saturation, competition binding, and kinetic experiments. These studies resulted in KD values of ∼3 nM, which is in good accordance with previously published data obtained from conventional receptor-ligand binding assays. The procedure described in this study simplifies classical binding studies to a kit-like assay. The receptors retained their binding properties even when preparations were dried completely. Transport and delivery of the material are conceivable without loss of biological activity. Therefore, other laboratories can perform binding studies without special equipment or the necessity to run a cell culture laboratory and/or to dissect tissue on their own.
Keywords: DPCPX; adenosine A1 receptor; low melting point agarose; radioligand; receptor kinetics; stably transfected cell lines.