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.
Our project will study a new and unexpected function of RIAM in regulating T cell immune responses. Our studies will provide new insights into mechanisms and pathways that regulate T cell activation. This knowledge may also identify new targets for therapeutic intervention for modulation of the immune responses either for suppression as in autoimmunity, allergy, asthma, graft rejection and graft versus host disease or for augmentation against pathogens and tumor antigens.
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