The role of intracellular trafficking pathways in calcium signaling


Principal Investigator: Suzette Farber-Katz
Abstract: DESCRIPTION (provided by applicant): Calcium signaling is essential for numerous biological processes. The NFAT transcription factors, which play key roles in immune cells, bone, heart and skeletal muscle, neurons, glia, pancreas and stem cells, are activated in response to Ca2+ mobilization. NFAT also plays crucial roles in developmental processes and malignant transformation and carcinogenesis. In non-excitable cells, NFAT transcription factors are activated by store-operated Ca2+ entry. Store depletion results in the activation of the ORAI plasma membrane Ca2+ channels by STIM1, a Ca2+ sensor in the endoplasmic reticulum. My sponsor's lab performed a genome-wide RNAi screen in HeLa cells to identify novel components of store-operated Ca2+ entry. The goal of this screen was to further understand the complex pathways associated with Ca2+ signaling. In addition, because current therapeutics target proteins involved in Ca2+ signaling (e.g., cyclosporin A, FK506), the hits from the RNAi screen could be studied as targets of future therapies. Here I propose to further characterize the regulation of Ca2+ signaling by studying four hits from our screen, UEV3, USP13, ArfGAP2, and TMP21, all of which have been implicated in intracellular trafficking. I chose to study these hits because I have previous experience studying proteins and pathways involved in trafficking. We hypothesize that trafficking mechanisms are involved in the movement of STIM1 from internal ER membranes to plasma membrane-apposed ER membranes. In Aim 1 of this proposal, I will capitalize on my strengths in cellular biology and trafficking by investigating the role of UEV3 and USP13 in Ca2+ signaling. I will identify post-translational modifications on STIM that are induced by UEV3/USP13 knockdown. I will also identify other proteins that might form a complex with UEV3 and USP13, and I will examine the localization of UEV3 and USP13 in relation to STIM1 and ORAI1. In Aim 2, I will perform similar experiments with ArfGAP2 and TMP21, two proteins that have been implicated in trafficking. Both proteins will be investigated for their ability to interact with and affect the function and localization of STIM and ORAI. In Ai 3, I will examine store- operated Ca2+ entry and biological function in cells from gene-targeted mice lacking or overexpressing selected trafficking hits from the screen. These experiments will provide me the opportunity to learn new skills and techniques associated with mouse genetic models and the study of primary cell types. The characterization of these proteins will lead to the elucidation of novel pathways that control calcium signaling.
Funding Period: 2013-04-01 - 2016-03-31
more information: NIH RePORT

Detail Information

Research Grants31

  1. Baylor Intellectual and Developmental Disabilities Research Center
    Huda Y Zoghbi; Fiscal Year: 2013
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  2. Intellectual and Development Disabilities Research Center
    Marc Yudkoff; Fiscal Year: 2013
    ..5 million from NICHD). The Center includes an excess of 70 Penn faculty at 15 departments at the Schools of Medicine, Veterinary Medicine, Nursing, the Wistar Institute, and the College of Arts and Sciences. ..
  3. Molecular Mechanisms of Ion Channels in T Lymphocytes
    Michael D Cahalan; Fiscal Year: 2013
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  4. Protein homeostasis mechanisms underlying enterovirus replication and evolution
    Raul Andino; Fiscal Year: 2013
    ..Core A: Administrative Core;and Core B: "High-throughput functional genomics and proteomics core. ..
  5. Toll-like receptor 9: trafficking and signaling
    Cynthia A Leifer; Fiscal Year: 2013
    ..This knowledge will provide the basis for the further development of CpG DNA as an adjuvant and therapeutic as well as develop mechanisms to interrupt the cycle of autoimmune pathology. ..
  6. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  7. Intestinal Disease-enterocyte toxin interaction
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    ..We have also developed the zebrafish for genetic studies and identified 13 families by forward screen as resistant to intoxication. The mutant genes in these families will be identified by positional-mapping and their function studied. ..
  8. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
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    BRUCE W STILLMAN; Fiscal Year: 2013
  10. Plasticity at the Excitatory Synapse
    Richard L Huganir; Fiscal Year: 2013
    ..Understanding these basic mechanisms of synaptic transmission and plasticity will provide insight into normal and abnormal brain function. ..
    Kenneth H Cowan; Fiscal Year: 2013
  12. A new class of immunomodulator, CRAC channel blockers
    Yousang Gwack; Fiscal Year: 2013
    ..Such small molecule blockers targeting Orai1 activity is likely to have much less side effects than immunosuppressive drugs such as cyclosporin A and tacrolimus because the CRAC channel is specifically predominant in immune cells. ..
  13. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..