Nearly all immune responses are the result of TCR signaling in the context of costimulation. Thus, theprocesses by which TCR and costimulatory signals are sensed and summed define distinct outcomes ofTCR engagement, including activation, anergy or death. An understanding of signal integration at thecellular level is essential if we are to understand the pathological consequences of these cellular outcomes,which include immunodeficiency and autoimmunity. While many of the proximal and distal events in T cellactivation are well studied, the molecular mechanisms underlying control of calcium entry, an essentialfeature of activation, are not fully understood. We will contribute to the goals of the Program by focusingspecifically on TFII-I, which is abundantly expressed in lymphoid cells and serves a prominent cytosolic function as a negative regulator of receptor-mediated calcium entry. Supression of calcium entry requirestwo sites within TFII-I that mediate binding to PLC-gamma: a tyrosine residue that supports phosphorylationdependentbinding and a neighboring interval that interacts with the PLC-gamma split PH domain. We foundthat TFII-I exerts its action by decreasing the density of cell-surface TRPC3 receptors. We propose thatTFII-I antagonizes calcium entry by interfering with the ability of PLC-gamma to promote transport of TRPC3channels to the plasma membrane. In a>human T cell line, removal of TFII-I results in a substantial increasein calcium influx upon T cell receptor engagement and upregulates expression of the inhibitory protein Spryl.To elucidate a role for TFII-I in regulating calcium entry in lymphocytes, we propose three aims: (1) to assessthe mechanism by which TFII-I modulates calcium entry in response to TCR engagement; (2) to examine theeffects of TFII-I ablation on antigen-specific T cell responses; and (3) to identify specific tyrosinephosphorylation pathways that regulate TFII-I activity. Modulation of calcium entry is likely to play a criticalrole in defining distinct outcomes of TCR engagement; consequently the proposed studies are highlysynergistic with all other components of the Program. Because anergizing signals are capable of uncouplingTCR engagement from calcium entry, determining how calcium entry is modulated by TFII-I may proveuseful in understanding how activated and anergic states are distinguished.
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