Store-operated Ca2+ entry (SOCE) is the process whereby loss of Ca2+ in the endoplasmic reticulum (ER) leads to Ca2+ entry into cells across the plasma membrane (PM). STIM1 and STIM2 are critical ER Ca2+ sensors that control the activation of Orai1, the store-operated Ca2+ channel. STIM1/2 are critical for T cell receptor (TCR)- mediated signaling and their deficiency in humans results in lymphoproliferative disorder and autoimmunity sug- gesting defects in T cell development and activation. In addition to ER Ca2+ release and SOCE, Ca2+ signals involve Ca2+ extrusion mechanisms. In lymphocytes, Plasma Membrane Ca2+/ATPase 4 (PMCA4) is the primary mediator of Ca2+ extrusion and is essential for normal function. Recently, we identified PMCA4 as a further target of STIM1 (but not STIM2) during lymphocyte activation. Unlike SOCE, STIM1-induced PMCA inhibition is initi- ated by Early Growth Response 1 (EGR1) and EGR4-mediated STIM1 upregulation. EGRs are ubiquitous and rapidly upregulated zinc finger transcription factors. The current proposal is based on the following hypothesis: TCR engagement stimulates EGR-mediated STIM1 upregulation in nave T cells that initiates Ca2+ clearance inhibition, critical for the maintenance of NFAT/NF-?B activation and subsequent T cell activation. This work is organized into 3 aims.
Aim 1 is to characterize EGR1/4-mediated control of T cell diversity and function. While EGR1KO mice exhibit previously described defects in early T cell development, preliminary findings re- ported here are the first to reveal a T cell phenotype in EGR4KO mice. We have observed altered progression through the double-negative stages, dysregulation of Ca2+ clearance during T cell activation and defective T cell expansion. These investigations will be expanded, both to include EGR1/4dKO mice and to include analysis of developmental, conventional and non-conventional subtypes.
Aim 2 is to assess how STIM1?597 knockin af- fects T cell activation. As established in our recent study, STIM1 truncation at amino acid 597 (STIM1?597) eliminates PMCA association without affecting Orai1 activation13. Preliminary analysis of T cells obtained from a recently generated STIM1?597 knock-in mouse reveals intriguing T cell subset-specific changes in Ca re- 2+ sponses. Implications to T cell activation will be determined by measuring transcription factor activation, cytokine expression patterns and T cell diversity.
Aim 3 is to define the mechanisms whereby POST coordinates functional STIM1-PMCA4 interactions. We revealed that EGR-induced STIM1 upregulation causes decreased PMCA function within the immunological synapse during T cell activation. However, a third protein termed POST interacts with both proteins and regulates Ca2+ clearance during store depletion. Our preliminary findings provide new insight into how these interactions are coordinated during T cell activation and define their functional impli- cations. The dynamics of these interactions will be determined using mutational analysis, FRET and Ca2+ en- try/clearance assays. Collectively, these studies reveal a novel new concept for how Ca2+ signals contribute to events that occur beyond the seconds to minutes time periods encompassing a single Ca2+ transient.
EGR-MEDIATED STIM1-PMCA EXPRESSION AND FUNCTION IN T CELL SUBSETS ? NARRATIVE STIM1-dependent Ca2+ signals represent the primary means of Ca2+ entry in T cells, with critical contributions to their activation. We have recently shown that, in addition to activating Ca2+ channels, STIM1 also inhibits the plasma membrane Ca2+ pump during T cell activation. The current proposal focuses on defining the molecular events that mediate this process and determining the impact of this new STIM1-mediated function on T cell- mediated immunity.
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