Binding of [125I] AB-MECA to the human cloned adenosine A3 receptor using the semliki forest virus expression system
✍ Scribed by M. Patel; C. Harris; K. Lundstrom
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 106 KB
- Volume
- 40
- Category
- Article
- ISSN
- 0272-4391
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✦ Synopsis
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 affinities 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.