Binding of [125I] AB-MECA to the human cloned adenosine A3 receptor using the semliki forest virus expression system

Abstract

The cDNA for the human adenosine A3 receptor was introduced into the pSFV1 vector, and the in vitro transcribed RNA was electroporated into baby hamster kidney (BHK) cells with pSFV-Helper RNA. This protocol resulted in packaging of a high titre Semliki Forest Virus (SFV)-A3 virus stock. Infection of confluent Chinese hamster ovary (CHO) cells with the SFV-A3 virus stock resulted in an expression of human adenosine A3 receptors that was twofold more than that obtained with usual transfection methods (as determined by radioligand binding studies). The binding of [125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-uronamide ([125I]AB-MECA) was specific and saturable (pKd = 8.8; Bmax = 0.5 pmol mg−1 protein). Adenosine receptor ligands were evaluated for their binding afffinities at the human cloned adenosine A3 receptor. The rank order of affinities of the ligands were: CGS 15943 > IB-MECA > APNEA > ligands with selectivity for adenosine A1, A2A, and A2B, receptors. However, the A1 selective ligand, GR79236 had little or no affinity for the human adenosine A3 receptor. In conclusion, the SFV expression system can be used to express the human cloned adenosine A3 receptor at high levels in CHO cells. This study has examined the binding affinities at the human cloned adenosine A3 receptor, of an extensive range of ligands for the adenosine family of receptors. Furthermore, CGS 15943 has been identified as a ligand with high affinity at the human cloned adenosine A3 receptor. Drug Dev. Res. 40:35–40, 1997. © 1997 Wiley-Liss, Inc.