P2Y2 nucleotide receptors are upregulated in vivo in response to tissue stress or injury where they mediate a variety of biological functions including mitogenesis and inflammation. These processes are intimately involved in proliferation, differentiation, development and apoptosis of cells. How do P2Y2 nucleotide receptors mediate these processes? The complete answer to this question will require knowledge of the signal transduction pathways utilized by P2Y2 nucleotide receptors and information regarding the method used by the receptor to link to these pathways. Preliminary data have already indicated some of these mechanisms. However, the focus of this study is to determine if components of the desensitization pathway of this receptor are involved in these events. The proposed experiments will utilize methods to interrupt the desensitization process of P2Y2 nucleotide receptors and mutations of the receptor that have altered desensitization properties.
The first aim of the project is to determine whether mutations of P2Y2 nucleotide receptors affect desensitization and subsequent activation of mitogenic and pro-inflammatory pathways. The ability of these mutant P2Y2 nucleotide receptors to link to stress- and mitogenic-activated protein kinases and/or mediate arachidonic acid release and eicosanoid production will be compared to wild-type receptors. These studies will be performed in a well-established cell line, 1321N1 human astrocytoma cells that lack endogenous P2Y2 receptors.
The second aim will introduce dominant-negative mutants of proteins involved in desensitization and antisense oligonucleotides to these proteins into 1321N1 cells expressing recombinant P2Y2 nucleotide receptors and a human breast cancer, estrogen receptor positive, cell line MDA-MB-231.
The third aim will introduce recombinant dominant-negative proteins into the cells under study.
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