Molecular Characterization of Purinergic Receptor and its Signal Transduction in the Vascular System

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It is well known that various purines exert diverse biological activities through P_1 and P_2 purinergic receptors in a variety of tissues. We attempted the molecular characterization of a purinergic receptor expressed in the vascular tissue and the signal transduction. Firstly, we cloned a full length rat P_1(A_1 subtype)receptor cDNA from rat liver lambdagt 10 cDNA library by hybridization screening using PCR-amplified dog A_1 receptor cDNA as a probe. Then, we screened rat aortic smooth muscle cDNA library by using rat A_1 receptor cDNA as a probe. However, we failed to detect a clone which represents P_2 receptors. We, then adapted the strategy of the expression cloning, and screened rat aortic smooth muscle cDNA library constructed in the expression vector pCDM8 by measuring the inositol phosphate production in Ltk^-cells transfected with subgroups of the pCDM8 cDNA library. We could not detect any library subgroup which confered a positive response. Finally, we tried molecular cloning of P_2 receptors by PCR using degenerate oligonucleotide primers corresponding to the 3rd and the 6th transmembrane domains which are well conserved among different G protein-coupled receptors. We isolated more than 40 clones which represented G protein-coupled receptors, among which A_1 receptor, ET_A endothelin receptor and alpha_1 and beta_1 adrenergic receptors were included. Two of them were considered to represent novel G protein-coupled receptors. We isolated full length clones of these two receptors. The transfection of either of the two cDNA clones into mammalian cells did not confer responsiveness to the P_2 ligand. We are now trying to find ligands for these two novel receptors.