One outcome of T-cell receptor (TCR) signaling is increased affinity and avidity of integrins for their ligands. This occurs through a process known as inside-out signaling, which has been shown to require several molecular components including the adapter proteins ADAP (adhesion and degranulation-promoting adapter protein) and SKAP-55 (55-kDa src kinase-associated phosphoprotein) and the small GTPase Rap1. Herein, we provide evidence linking ADAP and SKAP-55 to RIAM, a recently described adapter protein that binds selectively to active Rap1. We identified RIAM as a key component linking the ADAP/SKAP-55 module to the small GTPase Rap1, facilitating TCR-mediated integrin activation. We show that RIAM constitutively interacts with SKAP-55 in both a heterologous transfection system and primary T cells and map the region essential for this interaction. Additionally, we find that the SKAP-55/RIAM complex is essential both for TCR-mediated adhesion and for efficient conjugate formation between T cells and antigen-presenting cells. Mechanistic studies revealed that the ADAP/SKAP-55 module relocalized RIAM and Rap1 to the plasma membrane following TCR activation to facilitate integrin activation. These results describe for the first time a link between ADAP/SKAP-55 and the Rap1/RIAM complex and provide a potential new mechanism for TCR-mediated integrin activation.Integrins are cell surface receptors that mediate cell-cell interactions and cell-matrix interactions. These receptors are critical for T-cell migration to peripheral lymph nodes and inflammatory sites and are necessary for productive interactions between T cells and antigen-presenting cells (APCs) (22). The major integrins expressed on T cells are the ␤2 integrin LFA-1 (lymphocyte function-associated antigen 1; ␣L␤2) and the ␤1 family of integrins (very late antigen [VLA]; ␣4␤1, ␣5␤1, and ␣6␤1). LFA-1 binds to intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and ICAM-3, whereas the ␤1 family binds to vascular cell adhesion molecule 1 or the extracellular matrix protein fibronectin (12, 36). On resting T cells, integrins are maintained in an inactive state and adopt a lowaffinity conformation for their ligands. T-cell receptor (TCR) engagement by a peptide-major histocompatibility complex (MHC) complex induces integrin clustering as well as a conformational change leading to a higher-affinity state, thereby markedly enhancing the avidity of integrins for their ligands (1,22,32). This activation-induced modulation of integrin activation via antigen receptors (or other cell surface receptors) occurs through a process known as inside-out signaling (9, 22).The molecular basis for inside-out signaling remains poorly understood; however, several proteins in the TCR signaling pathway are known to be involved (22,39). Following engagement of the TCR and activation of src and syk family protein tyrosine kinases, the transmembrane adapter LAT (linker for activation of T cells) and the cytosolic adapter protein SLP-76 (76-kDa src homology 2 domain-containing leukocyt...

T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1–310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1–310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.

R e s e a R c h a R t i c l e1 0 2 0 jci.org Volume 125 Number 3 March 2015 therapeutic implications, since they can carry out graftversus-leukemia (GVL) responses with minimal GVHD. Results Generation and characterization of T cell-specific CRK T cells (data not shown).Western blotting of purified CD4 + T cells from Dko and WT mice revealed that levels of CRKI, CRKII, and CRKL in the mutant T cells were reduced by at least 95% ( Figure 1A and data not shown), and flow cytometric analysis confirmed loss of CRK protein expression (Figure 1, B and C).CRK/CRKL Dko mice were born at Mendelian ratios, and spleen, thymus, and lymph nodes exhibited normal cellularity (data not shown). Thymocyte populations were similar in WT and Dko mice, indicating that T cell development proceeded normally ( Figure 1D). Peripheral lymphoid organs showed no differences in proportions of CD4 + and CD8 + T cells, naive (CD62L hi CD44 lo ), memory (CD62L lo CD44 hi ), or activated (CD69 hi ) T cell subsets (Figure 1, D and E, and data not shown). Thus, these mice represented a suitable source of mature CRK/CRKL Dko T cells for functional studies. CRK/CRKL-deficient T cells show defects in adhesion and polarization.Since CRK proteins control adhesion in non-hematopoietic cells (4), we first asked whether integrin-dependent adhesion is defective in CRK/CRKL Dko T cells. On plates coated with ICAM-1, the ligand for the β 2 integrin LFA-1, WT preactivated CD4 + T cells showed about 10% basal binding, which was increased 2-to 5-fold after stimulation with the chemokines CXCL12 or CCL21, or anti-CD3 (Figure 2A). CRK/CRKL Dko T cells showed a significant reduction in adhesion to ICAM-1 under both basal and stimulated conditions. Indeed, chemokine stimulation induced almost no increased adhesion in these cells. Treatment with PMA bypassed the defect. This is most likely because the defect in CRK/CRKL Dko cells lies upstream of PKC signaling in the pathway leading to integrin activation, though PKC activation could also drive a parallel

T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.