ATP and ADP acting through P2-purinergic receptors are physiologically important extracellular signalling molecules in the cardiovascular system, in the central and peripheral nervous systems, and in many other tissues. Burnstock and Kennedy have proposed the existence of at least two P2-purinergic receptor subtypes (P2X- and P2Y-); other P2-purinergic receptor subtypes also apparently exist. As a group of signalling molecules, the P2- purinergic receptors are poorly understood. The availability of model systems for the study of these receptors is limited, and the lack of a P2-purinergic receptor antagonist has proven a liability. Recently, it has become clear that P2-purinergic receptors are members of a large group of cell surface proteins that regulate the inositol lipid/Ca2+ cascade, and it is now possible to associate at least the P2Y-purinergic receptor (P2Y-R) and another less well- defined P2-purinergic receptor with an important signalling mechanism. Our laboratory has adapted the turkey erythrocyte as a model to study receptor- and G-protein-regulated phospholipase C. We have observed that this cell expresses a P2Y-R, and we have taken advantage of this model to study the regulation of phospholipase C and its associated G-protein by P2Y-R, to study the properties of P2Y-R during agonist-induced desensitization, to develop a reversibly binding P2Y-R radioligand, and to identify a photolabel for these receptors. A long-term goal of this research is to define in depth the structural/functional specifics of the P2Y-R protein. A five year research plan has been organized around four specific aims. We will purify to homogeneity the turkey erythrocyte P2Y-R. The purified receptor will be used to delineate the properties and specificity of interaction of the P2Y-R with the phospholipase C-activating G-protein that we have recently purified from turkey erythrocytes. We will clone from a fetal turkey blood library the cDNA for the P2Y-R using either a protein sequence- based oligonucleotide probe or after polymerase chain reaction amplification of mRNA. The receptor will be transiently or stably expressed from the recombinant DNA using COS-7 cells, Chinese hamster ovary cells, and Sf9 insect cells using the baculovirus expression system. Low stringency hybridization using a probe derived from the turkey erythrocyte P2Y-R cDNA will be used to clone P2Y-R homologs from mammalian tissues such as rat astrocytes, bovine aortic endothelial cells, and rat C6-2B glioma cells. It is anticipated that this research will allow comparison of the P2Y-R to other G-protein-linked receptors and other adenine nucleotide binding proteins, will begin to place P2-purinergic receptor subclassification on firmer ground, and will put us in position to begin studies aimed at defining the structural components of P2- purinergic receptors necessary for their various signalling functions.
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