Extracellular ATP induces significant functional changes in many cell and tissue types. These responses are mediated by so-called P2-purinergic receptors which are activated upon binding ATP and other nucleotides. Recent physiological, biochemical, and pharmacological studies indicate that there are multiple ATP receptor subtypes: 1) ATP receptors which directly activate cation channels in some excitable cell types; 2) ATP receptors which elicit the rapid induction of non-selective membrane pores in certain immune effector cells; and 3) G-protein coupled ATP receptors which can activate inositol phospholipid-specific phospholipase C (PI-PLC) effector enzymes and Ca2+ mobilization. Agonist selectivity studies indicate that these G-protein coupled ATP receptors can be further sub- categorized into either of two broad nucleotide selectivity groups. ATP and ADP, but not non-adenine nucleotides, are equipotent in activating so- called P2y-purinergic receptors. Conversely, ATP and UTP, but not ADP, are equipotent in activating G-protein dependent PI-PLC and Ca2+ mobilization in certain cells. These ATP/UTP receptors appear to be expressed in an extremely wide range of human (and non-human) cell types including phagocytic leukocytes, epithelial cells in both the renal and respiratory tracts, and fibroblasts. This proposed project is directed towards the identification and structural/functional characterization of these ATP/UTP receptors by molecular biological and biochemical approaches. Four major groups of experiments are proposed. The first series will utilize previously designed expression cloning strategies to isolate the cDNA species which encode the functional ATP/UTP receptors expressed in the LH- 60 human leukocyte cell line. Similar expression cloning strategies have been utilized for cloning > 10 G-protein coupled receptors (GPCR) which, like the HL-60 cell ATP/UTP receptors, activate PI-PLC-based signal transduction. The proposed experiments will determine whether these ATP/UTP receptors can be classified within the growing superfamily of GPCR. In a second group of studies, hybridization probes (based on the cDNA encoding HL-60 cell ATP/UTP receptors), will be used to characterize expression of these, or related ATP/UTP receptor transcripts in various human and non-human cells/tissues known to be functionally responsive to these nucleotides. The various mechanisms by which these ATP/UTP receptors activate G-protein-dependent signal transduction will be examined in the third group of studies. Experiments will investigate the ability of ATP/UTP receptors to differentially activate G-protein dependent effector enzymes (with particular emphasis on phospholipases) in both intact cells and in broken cell systems. A fourth group of studies will utilize nucleotide photoaffinity probes and anti-peptide antibodies to identify intrinsic membrane proteins which are likely to constitute ATP/UTP receptor subtypes. Finally, studies will be initiated to generate mammalian cell lines transfected with cDNA encoding the cloned ATP/UTP receptors. These studies will facilitate on-going characterization of the physiological/pathological roles played by these, and related, ATP receptors in regulating signal transduction and biological action in diverse cells and tissues.
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