Abstract
CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.
Highlights
In the adaptive immune response, the physical interaction between a T cell and an antigen presenting cell (APC) leads to the polarization of the T cell, with accumulation of cell surface proteins and signaling molecules at the T cell–antigen-presenting cell (APC) contact site, forming a highly organized signaling platform known as the immunological synapse (IS) [1,2,3]
The elevated Ca2+ concentration is due to the IP3 binding to the IP3 receptors in the endoplasmic reticulum (ER) and the releasing the Ca2+, that is followed by the opening of CRAC (Ca2+ release activated Ca2+ channels) channels composed of the pore-forming subunit Orai1 and the ER-resident activator STIM1 [6,7,8]
It was described that Orai1 channels along with the STIM1 redistributed to the IS between a T cell and an APC [14]
Summary
In the adaptive immune response, the physical interaction between a T cell and an antigen presenting cell (APC) leads to the polarization of the T cell, with accumulation of cell surface proteins and signaling molecules at the T cell–APC contact site, forming a highly organized signaling platform known as the immunological synapse (IS) [1,2,3]. The encounter with APCs, where a specific antigen binding to the TCR/CD3 complex initiates different signaling cascades, results in T cell activation [4,5]. In this signaling event, the elevated Ca2+ concentration is due to the IP3 binding to the IP3 receptors in the endoplasmic reticulum (ER) and the releasing the Ca2+, that is followed by the opening of CRAC (Ca2+ release activated Ca2+ channels) channels composed of the pore-forming subunit Orai and the ER-resident activator STIM1 [6,7,8]. It has been demonstrated that expression of these ion channels, such as Kv1.3, KCa3.1 and Orai along with STIM1 is increased in activated human T cells, and they are involved in the Ca2+ signaling of T cells upon activation [10,12,13,14,15]
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