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Binding of [ 125 I] AB‐MECA to the human cloned adenosine A 3 receptor using the semliki forest virus expression system
Author(s) -
Patel M.,
Harris C.,
Lundstrom K.
Publication year - 1997
Publication title -
drug development research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.582
H-Index - 60
eISSN - 1098-2299
pISSN - 0272-4391
DOI - 10.1002/(sici)1098-2299(199701)40:1<35::aid-ddr3>3.0.co;2-u
Subject(s) - adenosine , adenosine a2b receptor , chinese hamster ovary cell , adenosine receptor , biology , semliki forest virus , adenosine a3 receptor , microbiology and biotechnology , receptor , adenosine a1 receptor , cgs 21680 , biochemistry , agonist , rna , gene
The cDNA for the human adenosine A 3 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)‐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 adenosine A 3 receptors that was twofold more than that obtained with usual transfection methods (as determined by radioligand binding studies). The binding of [ 125 I]N 6 ‐(4‐amino‐3‐iodobenzyl)adenosine‐5′‐N‐methyl‐uronamide ([ 125 I]AB‐MECA) was specific and saturable (pK d = 8.8; B max = 0.5 pmol mg −1 protein). Adenosine receptor ligands were evaluated for their binding afffinities at the human cloned adenosine A 3 receptor. The rank order of affinities of the ligands were: CGS 15943 > IB‐MECA > APNEA > ligands with selectivity for adenosine A 1 , A 2A , and A 2B , receptors. However, the A 1 selective ligand, GR79236 had little or no affinity for the human adenosine A 3 receptor. In conclusion, 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 examined the binding affinities at the human cloned adenosine A 3 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 A 3 receptor. Drug Dev. Res. 40:35–40, 1997. © 1997 Wiley‐Liss, Inc.