Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated done (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5'- and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P-2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta,gamma-methylene ATP and alpha,beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximate to uridine triphosphate > ADP approximate to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P-2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells. The cloned receptor is indeed expressed in rat pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P-2U purinoceptor. These findings suggest that the P-2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.