One of the principal mechanisms by which HIV causes acquired immune deficiency is by suppressing T lymphocyte immune responsiveness. This appears to be mediated at least in part by binding of the HIV envelope protein gp120 to its T cell receptor CD4 initiating processes which render T cells unresponsive to antigen. We propose to test the hypothesis that gp120-CD4 interaction triggers transduction of signals which cause """"""""desensitization"""""""" of T cell antigen receptors (TCR). We will further test the hypothesis that desensitization is mediated via activation of protein kinases which exert their efforts by phosphorylation of specific substrate targets in the TCR CD3 complex. We will first (aim 1) assess the affect of CD4 ligation on various early events in TCR mediated T cell activation. This will allow us to test the basic hypothesis and to define the molecular site at which the transduction cascade initiated by ligation of CD4 inhibits signaling through TCR. We will then define, using specific peptide substrates and inhibitors of kinases, the protein kinases which are activated following gp120-CD4 interaction and determine if their activation is causally related to TCR desensitization (aim 2). Results of aim 2 should implicate the phosphorylation of certain CD3 components at serine/threonine or tyrosine residues with specific flanking amino acid sequences in mediating desensitization. Based on this information, we will generate cells bearing mutant receptors which lack the implicated phosphorylation sites and use these to define the role of specific phosphorylation events in desensitization. These experiments will employ human (h) and mouse T cells, T lymphomas, hCD4 transfected mouse T hybridomas and hCD4 transgenic mice in conjunction with a number of experimental methods including biochemical analysis of inositol lipid metabolism, [Ca2+]i mobilization, protein phosphorylation, protein kinase activation, and activation of gene expression, as well as assays of IL2 secretion and T cell proliferation. Recombinant DNA technology and transfection will be utilized to create cells which express mutant CD3 components. The proposed studies should define the molecular basis of gp120 induced suppression of T cell activation and provide critical insight for eventual development of approaches to pharmacologic intervention to prevent or reverse immunosuppression in AIDS.
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