The role of RIAM in T cell immunity and tolerance

描述（由申请人提供）：本项目的目标是研究RIAM（MRL家族的衔接分子）在T细胞应答中的作用。我们确定和分子克隆RIAM（Rap 1GTP相互作用衔接子分子），通过其与活性，GTP结合Rap 1。RIAM包含N-末端卷曲螺旋区、中心Ras缔合（RA）和普列克底物蛋白同源性（PH）结构域以及富含脯氨酸的C-末端区，该C-末端区具有与肌动蛋白调节蛋白Ena/VASP的EVH 1结构域相互作用的多个FPPP基序和与profilin相互作用的多个XPPPP基序。通过与Ena/VASP蛋白的相互作用，RIAM起到细胞骨架调节剂的作用。RIAM也参与激活？1、？T细胞中的2种整合素？二？3血小板整合素。我们确定RIAM在TCR介导的信号传导中具有意想不到的作用。RIAM直接与PLC交互-？1，并参与肌醇1，4，5-三磷酸（IP 3）的产生，细胞内钙离子的动员，核转位的NFAT和Ras的激活。内源性RIAM的消除消除了这些事件，尽管ZAP-70的磷酸化和SLP-76-LAT信号体的形成仍然不受影响。我们确定，PH结构域的RIAM具有特异性的PLC-？1底物，PI（4，5）P2，而RA结构域结合Rap 1只在质膜，而不是在内膜室。RA和PH结构域都对其天然配偶体具有低亲和力结合。引人注目的是，RA和PH结构域被整合到一个单一的结构单元中，RA和PH组分与其天然伴侣的结合是RIAM向质膜募集所需的。RIAM还与Gads相互作用，并在其PH结构域中经历Lck介导的磷酸化。我们的研究结果揭示了一个意想不到的作用RIAM作为第二个和不可或缺的节点的信号整合是共同需要与SLP-76-LAT信号体激活PLC-？1.通过RIAM调节PLC-？1激活仍不清楚。目前还不清楚RIAM是如何与其他信号分子整合在信号组件中的，以及RIAM对PLC-？1的活化将影响肌动蛋白重组和整合素亲合力调节，从而影响抗原相遇期间的T细胞相互作用动力学和功能结果。为了解决这些问题，我们将致力于本提案中所述的具体目标。具体目标1：研究RIAM如何调控PLC-？1.具体目标2：确定RIAM如何调节信号微簇的形成、分布和信号输出。具体目标3：研究RIAM在抗原刺激过程中如何调节原代T细胞TCR信号传导、肌动蛋白动力学、整合素亲合力调节和相互作用动力学。

DESCRIPTION (provided by applicant): The goal of the present project is to study the role of RIAM, an adaptor molecule of the MRL family, in T cell responses. We identified and molecularly cloned RIAM (Rap1GTP-interacting adaptor molecule) through its association with active, GTP-bound Rap1. RIAM contains an N-terminal coiled-coil region, central Ras association (RA) and pleckstrin homology (PH) domains, and a proline-rich C-terminal region with multiple FPPPP motifs interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP and multiple XPPPP motifs interacting with profilin. Through its interaction with Ena/VASP proteins RIAM functions as cytoskeletal regulator. RIAM is also implicated in activation of ?1 and ?2 integrins in T cells ?II?3 integrin in platelets. We determined that RIAM has an unexpected role in TCR-mediated signaling. RIAM interacts directly with PLC-?1 and is involved in the generation of inositole 1,4,5-trisphosphate (IP3), mobilization of intracellular calcium ions, nuclear translocation of NFAT and activation of Ras. Elimination of endogenous RIAM abrogated these events, although phosphorylation of ZAP-70 and formation of the SLP-76-LAT signalosome remained unaffected. We determined that the PH domain of RIAM has specificity for the PLC-?1 substrate, PI(4,5)P2, whereas the RA domain binds Rap1 only at the plasma membrane and not in the endomembrane compartments. Both the RA and PH domains have low affinity binding for their natural partners. Strikingly, the RA and PH domains are integrated into a single structural unit and binding of both RA and PH components to their natural partners is required for recruitment of RIAM to the plasma membrane. RIAM also interacts with Gads and undergoes Lck-mediated phosphorylation in its PH domain. Our results reveal an unexpected role of RIAM as a second and indispensable node of signal integration that is co-required with the SLP-76-LAT signalosome for activation of PLC-?1. The mechanisms via which RIAM regulates PLC-?1 activation remain unclear. It is also unclear how RIAM is integrated in signaling assemblies with other signaling molecules and whether the effects of RIAM on PLC-?1 activation would impact on actin reorganization and integrin avidity modulation thereby affecting T cell interaction dynamics and functional outcomes during antigen encounter. To address these questions we will undertake the Specific Aims described in the present proposal. Specific Aim 1: To dissect how RIAM regulates activation of PLC-?1. Specific Aim 2: To determine how RIAM regulates formation, distribution and signal output from signaling microclusters. Specific Aim 3: To examine how RIAM regulates TCR signaling, actin dynamics, integrin avidity modulation and interaction dynamics of primary T cells during stimulation by antigen.