BINDING OF [I-125] AB-MECA TO THE HUMAN CLONED ADENOSINE A(3) RECEPTOR USING THE SEMLIKI-FOREST-VIRUS EXPRESSION SYSTEM

The cDNA for the human adenosine A(3) receptor was introduced into the pSFV1 vector, and the in vitro transcribed RNA was electroporated int o baby hamster kidney (BHK) cells with pSFV-Helper RNA. This protocol resulted in packaging of a high titre Semliki Forest Virus (SFV)-A(3) virus stock. Infection of confluent Chinese hamster ovary (CHO) cells with the SFV-A(3) virus stock resulted in an expression of human adeno sine Ag receptors that was twofold more than that obtained with usual transfection methods (as determined by radioligand binding studies). T he binding of [I-125]N-6-(4-amino-3-iodobenzyl)adenosine- 5'-N-methyl- uronamide ([I-125]AB-MECA) was specific and saturable (pK(d) = 8.8; B- max = 0.5 pmol mg(-1) protein). Adenosine receptor ligands were evalua ted for their binding affinities at the human cloned adenosine A(3) re ceptor. The rank order of affinities of the ligands were: CCS 15943 > IB-MECA > APNEA > ligands with selectivity for adenosine A(1), A(2A), and A(2B), receptors. However, the Al selective ligand, GR79236 had li ttle or no affinity for the human adenosine A(3) receptor. In conclusi on, the SFV expression system can be used to express the human cloned adenosine A(3) receptor at high levels in CHO cells. This study has ex amined the binding affinities at the human cloned adenosine A(3) recep tor, of an extensive range of ligands for the adenosine family of rece ptors. Furthermore, CGS 15943 has been identified as a ligand with hig h affinity at the human cloned adenosine A(3) receptor. (C) 1997 Wiley -Liss, Inc.