Ca2+ influx though CRAC (Ca2+ release activated Ca2+) channels is critical for immune cell functions. Stimulation through receptors depletes intracellular Ca2+ stores and subsequently leads to opening of CRAC channels on the plasma membrane. Recently, Orai1 (CRACM1) and STIM1 were identified as a pore component of the CRAC channel and a mediator between Ca2+ store depletion and CRAC channel opening, respectively. However, much remains to be understood about the molecular composition and the mechanism of CRAC channel regulation. We have used large scale affinity protein purification under store depleted conditions to identify regulators of the CRAC channel. We have identified CRACR2A (Ca2+ release activated Ca2+ channel Regulator 2A) as a novel regulator of CRAC channels using this approach.
The Specific Aims are: (1) To test the hypothesis that Orai1-CRACR2A interaction is critical for the CRAC channel function. The activities of Orai1 mutants incapable of binding to CRACR2A will be analyzed in Orai1-null primary cells. (2) To test the hypothesis that CRACR2A stabilizes the Orai1-STIM1 complex. Our preliminary data suggest that CRACR2A can directly interact with both Orai1 and STIM1. The functions of Orai1 and STIM1 will be examined using CRACR2A knock-down cells. The functional importance of reciprocal interaction of Orai1, STIM1, and CRACR2A will be determined. (3) To test the hypothesis that CRACR2A is critical for the T cell function. Knock-down of CRACR2A in Jurkat T cells decreased Ca2+ entry. We will conditionally target the CRACR2A gene to determine the role of CRACR2A in store-operated Ca2+ entry in peripheral T cells. The experiments on the novel regulator of the CRAC channel are timely because of the very recent discovery of Orai1 and STIM1. In the short term, the proposed experiments should provide new molecular probes for investigating the regulatory mechanism of store-operated Ca2+ entry in mammalian cells, particularly in immune cells. In the long term, we will test the possibility of development of drugs to modulate the function of the CRAC channel;thereby immune activation. Public Health Relevance: We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.

Public Health Relevance

Statement We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083432-03
Application #
8098129
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Mallia, Conrad M
Project Start
2009-07-01
Project End
2014-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
3
Fiscal Year
2011
Total Cost
$377,339
Indirect Cost
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Choi, Hyewon; Srikanth, Sonal; Atti, Elisa et al. (2018) Deletion of Orai1 leads to bone loss aggravated with aging and impairs function of osteoblast lineage cells. Bone Rep 8:147-155
Woo, Jin Seok; Srikanth, Sonal; Kim, Kyun-Do et al. (2018) CRACR2A-Mediated TCR Signaling Promotes Local Effector Th1 and Th17 Responses. J Immunol 201:1174-1185
Sen, Subha; Wang, Fei; Zhang, Jing et al. (2018) SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate ROR?t activity in a PKC-?-dependent manner. Proc Natl Acad Sci U S A 115:E458-E467
Choi, Dongwon; Park, Eunkyung; Jung, Eunson et al. (2017) Laminar flow downregulates Notch activity to promote lymphatic sprouting. J Clin Invest 127:1225-1240
Srikanth, Sonal; Woo, Jin Seok; Gwack, Yousang (2017) A large Rab GTPase family in a small GTPase world. Small GTPases 8:43-48
He, Zhiheng; Wang, Fei; Zhang, Jing et al. (2017) Regulation of Th17 Differentiation by IKK?-Dependent and -Independent Phosphorylation of ROR?t. J Immunol 199:955-964
Srikanth, Sonal; Woo, Jin Seok; Sun, Zuoming et al. (2017) Immunological Disorders: Regulation of Ca2+ Signaling in T Lymphocytes. Adv Exp Med Biol 993:397-424
Lee, Sung Hee; Park, Yongtae; Song, Minju et al. (2016) Orai1 mediates osteogenic differentiation via BMP signaling pathway in bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 473:1309-1314
Srikanth, Sonal; Kim, Kyun-Do; Gao, Yuanyuan et al. (2016) A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals. Sci Signal 9:ra31
He, Zhiheng; Wang, Fei; Ma, Jian et al. (2016) Ubiquitination of ROR?t at Lysine 446 Limits Th17 Differentiation by Controlling Coactivator Recruitment. J Immunol 197:1148-58

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