Chemokine receptors CXCR4 and CCR5 play critical roles in H1V infection. Our preliminary studies and work from other laboratories indicate that binding of the envelope protein gp 120 with CXCR4 or CCR5 in T cells generates cytoplasmic signals that induce chemotaxis. In this revised application, we focus on how HIV gp 120 binding to chemokine receptors may trigger signal transduction and alter T-cell chemotaxis. Given the importance of HIV gp120 in immune regulation and the development of AIDS, understanding how signaling molecules control changes in chemotaxis is of fundamental importance. H1V gp120-induced inappropriate chemotaxis could lead to enhanced migration of T cells to anatomical sites of virus production. We have shown that key regulatory signaling molecules linked to CXCR4 and CCR5 activation are protein tyrosine kinases, components of focal adhesions, P13-kinase (P13-K) and nitric oxide (NO). The objectives of this proposal are to elucidate both the normal signaling pathways mediated by cognate ligand, stromal cell-derived factor 1 a (SDF 1 a) and macrophage inflammatory protein 1 b (MW I b) binding to the CXCR4 or CCR5 receptor respectively and subversion of signaling by HIV gp 120. The first specific aim of this proposal is to characterize which components of the protein tyrosine kinase (PTK) pathway, such as Src-related kinases, phosphatase SHP1/SHP2 and focal adhesion kinase RAFTKIPyk2, are required for the receipt of signals from CXCR4 or CCR5 upon gp 120 or cognate ligand binding.
The second aim i s to elucidate functions of the downstream signaling molecules such as P13K/AKT, NO and NF kappa B (NF-kB) that receive signals from PTKs. This will be achieved by using specific pharmacological inhibitors as well as wild type and dominant negative mutant constructs. The activity of these signaling molecules in response to natural ligands (e.g., chemokines) vs. HIV gp120 will be assessed, as well as changes in, including regulation of subcellular localization, post-translational modification and association with novel regulatory proteins. In each case, we seek to determine the importance of these molecules in mediating chemotaxis. Elucidation of these signaling pathways should provide insights into the molecular mechanisms whereby chemokine receptors CXCR4 or CCR5 can participate in inappropriate chemotaxis following HIV infection. These studies should assist in the development of novel strategies to protect or augment immune integrity in people with HIV disease.
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