The primary role of B cells, antibody production and antigen elimination, is controlled by the B cell receptor (BCR) complex. BCR crosslinking activates a recently discovered and powerful signaling system mediated by the ER membrane proteins, STIM1 and STIM2. Interacting directly with the PM, STIM proteins expose a reactive domain that avidly binds and traps a specialized family of channel proteins, Orai1, Orai2, and Orai3. The Orai1 channels are exceedingly selective Ca2+ channels that, upon direct binding to STIM sensors, become activated to conduct Ca2+ ions into the junctional cytosolic space. This highly controlled entry of Ca2+ is crucial for two reasons: (i) to replenish Ca2+ within the ER preventing cell stress from protein misfolding, and allowing Ca2+ release signals to be maintained;(ii) to provide longer term and spatially defined Ca2+ signals mediating control over transcription, growth, or apoptosis. The STIM-Orai signaling pathway has particular significance in B cells - the precise coordination of Ca2+ release and entry signals mediates oscillatory Ca2+ signals, the amplitude and duration of which determine how B cells respond to BCR antigen-binding to undergo either proliferation, anergy, or apoptosis. The work combines molecular, biophysical, and cellular approaches to study STIM and Orai proteins using the DT40 B cell line and HEK293 human kidney-derived cells. DT40 B cells retain functional BCR-coupled signaling machinery and we have lines in which each STIM and Orai protein is knocked out. Using these cells our three specific aims are: 1. To examine the distinct functional roles of STIM1 and STIM2 proteins in mediating Ca2+ entry signals. 2. To ascertain how STIM1 and STIM2 proteins interact with and control Orai Ca2+ channels. 3: To examine the STIM-Orai Ca2+ signaling microenvironment in B cells. The studies dissect a novel Ca2+ signaling process fundamentally connected to the BCR-coupled machinery, exerting crucial regulatory control over B cell function. The STIM-Orai signaling pathway and its central role in Ca2+ signal generation in B cells provides a novel and important pharmacological target. The size and duration of Ca2+ signals are primary determinants of B cell fate in response to BCR activ- ation - cell division, maintenance, or, in the case of self-recognition, cell death. Defining the mechanistic operation of this long-acting Ca2+ signaling pathway and examining its pharmacological modification by the borate, 2-APB, provides a target through which B cell function and development can be modified providing the potential to control major immunological diseases including primary B cell deficiencies, lymphoproliferative disorders such as chronic lymphocytic leukemia, and autoimmune diseases.

Public Health Relevance

B cells, the crucial blood cells that produce antibodies and provide immunity, are fundamentally controlled by calcium signals generated by the binding of antigens to the B cell surface. An entirely new signaling pathway involving important regulatory proteins that move throughout the cell, trigger calcium signals that cause control of key B cell functions including growth and differentiation. These new signaling pathways provide vital new targets to pharmacologically control B cell function and development, providing the means to control major immunological diseases including lymphoproliferative disorders such as chronic lymphocytic leukemia and autoimmune diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI058173-11
Application #
8668881
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Ferguson, Stacy E
Project Start
2004-01-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
11
Fiscal Year
2014
Total Cost
$378,675
Indirect Cost
$131,175
Name
Pennsylvania State University
Department
Physiology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
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Zhou, Yandong; Wang, Xizhuo; Wang, Xianming et al. (2015) STIM1 dimers undergo unimolecular coupling to activate Orai1 channels. Nat Commun 6:8395
Wang, Xizhuo; Wang, Youjun; Zhou, Yandong et al. (2014) Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site. Nat Commun 5:3183
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