There has been increasing interest in a large family of very widely distributed purinergic receptors. We are attempting currently to express the cDNA of A2 and putative A1 adenosine receptor genes in COS cells. A remarkable feature of A1 receptors is their property of interacting with several different effector systems in various tissues. Hypothesis 1 is that adenosine A1 and A2 receptors are composites of multiple receptor subfamilies that have different tissue distributions. We will clone putative subtypes of A1 and A2 receptors and will determine the tissue distribution of mRNA's by in situ hybridization. We will transfect COS and CHO cells to express individual subtypes of adenosine receptors. Such cells will be useful for screening new subtype-selective drugs. We will attempt to produce receptor subtype-selective antisera for use in Western blots. Hypothesis 2a is that the same A1 receptors are capable of interacting with several different G-proteins. We will use immunoblots to identify G proteins that are copurified by agonist affinity chromatography with A1 receptors from brain and adipose tissue. We have identified GH4 pituitary cells as a tissue culture line that has three different effector responses to A1 receptor activation (adenylyl cyclase, K+ channel conductance, and phospholipase C). Hypothesis 2b is that A1 receptors and various G proteins of GH4 cells become uncoupled at different rates during receptor desensitization. Hypothesis 2c is that coupling of various G proteins to A1 receptors of GH4 cells are differentially sensitive to pertussis toxin. To learn what G proteins produce the various A1-receptor mediated effector responses, the time courses of: i) changes in adenosine- mediated effector responses; ii) the amounts of various G-proteins coupled to A1 receptors; and iii) other receptor and G protein characteristics; will be measured during desensitization and following exposure to graded doses of pertussis toxin. Hypothesis 3a is that myristylation of G protein alpha-subunits and the nature of G protein beta and gamma subunits influences coupling of G proteins to A1 adenosine receptors. Hypothesis 3b is that A1 receptor subtypes couple to various G proteins with differential selectivities. Reconstitution of receptor-G protein coupling will be assessed using homogeneous recombinant G-proteins expressed in insect cells, and either native or recombinant individual receptor subtypes. Hypothesis 4 is that it will be possible to clone P2(ATP) receptors through the use of degenerate primers corresponding to conserved sequences of adenosine receptors and other G-protein interactive receptors. The polymerase chain reaction will be used to amplify cDNA derived by reverse transcription of mRNA prepared from tissues known to be rich in individual P2(ATP) receptor subtypes: megakaryocytes, P21; smooth muscle cells, P2X; and endothelial cells, P2Y. Individual cDNA's selected on the basis of their tissue distribution will be cloned and sequenced. Full length clones of candidate receptors will be obtained from appropriate cDNA libraries and transfected into COS cells. The cells will be screened with P2 selective ligands. The long term objectives of this project are to learn more about the molecular mechanisms that underlie the myriad receptor-mediated modulatory actions of purines, and to produce cell lines that express recombinant receptors for use in the identification of receptor subtype- selective drugs.
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