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，一种MRL家族的适配分子，在T细胞反应中的作用。我们通过与活性的、GTP结合的RAP1分子结合，鉴定并分子克隆了RIAM(Rap1GTP相互作用接头分子)。RIAM含有一个N-末端卷曲螺旋结构域、中央RAS结合(RA)和Pleckstrin同源(PH)结构域，以及一个富含Pro的C-末端区域，其中多个FPPPP基序与肌动蛋白调节蛋白Ena/Vasp的EVH1结构域相互作用，以及多个XPPPP基序与Profilin相互作用。RIAM通过与Ena/Vasp蛋白相互作用，发挥细胞骨架调节作用。RiAM还参与激活T细胞中的整合素β1和整合素2、血小板中的整合素II 3。我们确定RIAM在TCR介导的信号转导中具有意想不到的作用。RIAM直接与PLC-1相互作用，参与1，4，5-三磷酸肌醇(IP3)的生成、细胞内钙离子的动员、NFAT的核转位和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信号体的形成仍然没有受到影响。我们确定RIAM的PH结构域具有PLC-1底物PI(4，5)P2的特异性，而RA结构域仅与质膜结合，而不与内膜室结合。RA和PH结构域与其天然伴侣的亲和力都很低。值得注意的是，RA和PH结构域被整合到一个单一的结构单元中，RA和PH成分都需要与它们的天然伙伴结合，才能将RIAM招募到质膜上。RIAM还与Gads相互作用，并在其PH域经历Lck介导的磷酸化。我们的结果揭示了RIAM作为第二个和不可或缺的信号整合节点的意外作用，这是与SLP-76-LAT信号小体共同激活PLC-1所必需的。RIAM调节PLC-1激活的机制尚不清楚。目前还不清楚RIAM如何与其他信号分子整合在信号集合体中，以及RIAM对PLC-1激活的影响是否会影响肌动蛋白重组和整合素亲和力的调节，从而影响T细胞在抗原相遇时的相互作用动力学和功能结果。为了解决这些问题，我们将采取本提案中所述的具体目标。具体目标1：剖析RIAM如何调节PLC的激活？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。具体目的3：研究RIAM在抗原刺激下对原代T细胞TCR信号、肌动蛋白动力学、整合素亲和力调节和相互作用动力学的调节作用。

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.