Nearly all immune responses are the result of TCR signaling in the context of costimulation. Thus, the processes by which TCR and costimulatory signals are sensed and summed define distinct outcomes of TCR engagement, including activation, anergy or death. An understanding of signal integration at the cellular 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 cell activation are well studied, the molecular mechanisms underlying control of calcium entry, an essential feature of activation, are not fully understood. We will contribute to the goals of the Program by focusing specifically 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 requires two sites within TFII-I that mediate binding to PLC-gamma: a tyrosine residue that supports phosphorylationdependent binding and a neighboring interval that interacts with the PLC-gamma split PH domain. We found that TFII-I exerts its action by decreasing the density of cell-surface TRPC3 receptors. We propose that TFII-I antagonizes calcium entry by interfering with the ability of PLC-gamma to promote transport of TRPC3 channels to the plasma membrane. In a>human T cell line, removal of TFII-I results in a substantial increase in 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 assess the mechanism by which TFII-I modulates calcium entry in response to TCR engagement;(2) to examine the effects of TFII-I ablation on antigen-specific T cell responses;and (3) to identify specific tyrosine phosphorylation pathways that regulate TFII-I activity. Modulation of calcium entry is likely to play a critical role in defining distinct outcomes of TCR engagement;consequently the proposed studies are highly synergistic with all other components of the Program. Because anergizing signals are capable of uncoupling TCR engagement from calcium entry, determining how calcium entry is modulated by TFII-I may prove useful in understanding how activated and anergic states are distinguished.
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