Nucleotides released during ischemia or mechanical injury to nervous tissue activate P2Y purinergic receptors that act synergistically with growth factors to stimulate astrocyte proliferation, astrogliosis, and the formation of """"""""glial scarring"""""""". Astrocytic P2Y2 purinergic receptors couple to mitogen-activated protein kinases ERKI/2, phospholipase C-dependent mobilization of intraceUular calcium, activation of focal adhesion kinase (FAK), c-Src kinase, and other signaling events. Membrane microdomains, and caveolae, are believed to serve as scaffolds that facilitate protein-protein interactions among signaling proteins, thus integrating pathways. The fundamental hypothesis of this proposal is that P2Y2 receptor localization in membrane microdomains, e.g., caveolae, lipid rafts, and clathrin-coated vesicles, underlies coupling to and uncoupling from purinergic activated signaling pathways.
Four specific aims will be undertaken in well-established astrocytic model systems including a cell line stably transfected with recombinant P2Y2 receptor.
Specific Aim 1 : Determine the location of the P2Yz receptor and transducers within membrane microdomains during purinergic signaling events in astroglial cells. These studies will involve three independent approaches: indirect immunofluorescence analysis using laser scanning confocal microscopy, isolation of discrete membrane microdomains using subcellular fractionation procedures, and immunoprecipitation assays. We postulate that P2Y2 receptor desensitization, resensitization and sequestration are pharmacodynamic events functionally linked to receptor trafficking between specific membrane microdomains, and thus, we will undertake a kinetic analysis of the subcellular trafficking or translocation of P2Y2 receptors.
Specific Aim 2 : To test the hypothesis that structural motifs of the P2Y2 receptor determine its location within membrane microdomains (caveolae/lipid rafts) and coupling to specific signaling pathways. The role of structural domains in the P2Y2 receptor, including the integrin-binding RGD (arginine-glycine-aspartate), a putative caveolin-binding motifs (fXfXXXXf), where f represents aromatic amino acids, X is any amino acid) and determinants in the C-terminal domain essential for receptor internalization, will also be evaluated for their impact on receptor co-localization and interaction with signaling molecules and caveolae membrane microdomains.
Specific Aim 3 : To test the hypothesis that membrane microdomain scaffolding molecules, caveolins and beta-arrestins, affect P2Y receptor coupling to signaling pathways. We will also examine the effects of cholesterol-depleting drugs (filipin III complex, nystatin and cyclodextrins) and inhibitors of clathrin-mediated endocytosis (phenyl arsenate and hypertonic media) on P2Y2 receptors signaling and trafficking using the three complementary experimental approaches outlined in Specific Aim 1.
Specific Aim 4 : To test the hypothesis that P2Yz receptor phosphorylation/dephosphorylation modulate receptor interactions underlying signaling and translocation. Experiments will be designed to determine if there is a functional correlation between P2Y2 receptor phosphorylation and its translocation from specific membrane microdomains. Insights gained into the mechanisms of purinergic receptor signaling in astrocytic cells and the role of extracellular nucleotides in neurodegenerative processes may provide novel targets for therapeutic strategies in the management of brain injury, synaptogenesis, as well as other neurological conditions.
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