Rap1A-deficient T and B cells show impaired integrin-mediated cell adhesion (original) (raw)
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The Small GTPase, Rap1, Mediates CD31-induced Integrin Adhesion
Journal of Cell Biology, 2000
Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via  1 (VLA-4) and  2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhe-sion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Rasproximal signaling, by which Rap1 might mediate transformation reversion.
The role of Rap1 in integrin-mediated cell adhesion
Biochemical Society Transactions, 2001
Rap1 is a member of the Ras-like small GTPases. Originally the protein was identified in a genome-wide screen for suppressors of Ras transformation, but the mechanism of this reversion remained elusive. We have investigated the signalling function of Rap1. We observed that Rap1 is activated by a large variety of stimuli, including growth factors, neurotransmitters and cytokines. Common second messengers like cAMP, diacylglycerol and calcium are mediators of this activation. These messengers activate guanine nucleotide exchange factors (GEFs), the most notable of which is Epac (exchange protein directly activated by cAMP). However, the downstream effectors of Rap1 are less clear. Although direct connections of Rap1 with the serine/threonine kinases Raf1 and B-raf have been reported, we were unable to find functional evidence for an interaction of endogenous Rap1 signalling with the Raf/extracellular-signal-regulated kinase (ERK) pathway. Instead we observe a clear connection of Rap1 with inside-out signalling to integrins. Indeed, introduction of a constitutively active Rap1 as well as Epac induces integrin-mediated cell adhesion, whereas inhibition of Rap1 signalling by the introduction of Rap1GAP (GTPase-activating protein) inhibits inside-out activation of integrins. More importantly, activation of a G s -protein-coupled receptor results in integrinmediated cell adhesion, by a pathway involving Epac and Rap1. From these results, we conclude that one of the functions of receptor-induced Rap1 activation is inside-out regulation of integrins.
Immunology Letters, 2004
Dynamic regulation of integrin-mediated adhesion is central to lymphocyte trafficking and antigen recognition. The small GTPase Rap1 is a potent stimulator of leukocyte integrins through modulation of affinity and avidity. In addition, lymphocyte Rap1 has unique abilities to trigger cell polarization and enhance cell motility. These characteristics of Rap1 contribute to adhesive interactions with antigen-presenting cells (APC) and the vascular endothelium. In the process of elucidating the molecular mechanisms of Rap1-mediated integrin activation, we have identified a novel Rap1-binding molecule, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL). RAPL is predominantly expressed in immune cells, and mediates Rap1-triggered integrin activation upon TCR engagement and chemokine stimulation. Importantly, Rap1/RAPL signaling cooperatively regulates cell polarization and integrin activation. The linkage between cell polarization and integrin activation through Rap1/RAPL signaling likely provides immune cells with their dynamic trafficking capability.
Journal of Biological Chemistry, 2002
In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (␣ L  2) and VLA-4 (␣ 4  1). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GT-Pase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn 2؉-and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn 2؉-and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn 2؉ , KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTPbound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn 2؉-and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.
Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion
The Journal of Cell Biology, 2004
ap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that R this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases.
Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin 4 1
Journal of Leukocyte Biology, 2007
The ␣41 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that ␣41 and CCR9 are coexpressed mainly on double-and single-positive thymocytes and that CCL25 strongly stimulates CD4 ؉ CD8 ؉ and CD4 ؉ CD8adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9 ؉ /␣41 ؉ human T cell line Molt-4. To study the role of CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTPinteracting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and adapter protein that binds selectively to active Rap1 (RIAM), we knocked down their expression and tested transfectant attachment to ␣41 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial ␣41-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by ␣41 could contribute to control their trafficking in the thymus during maturation and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in insideout signaling, leading to stimulated adhesion.
Blood, 2007
Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3+ human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1α (SDF-1α)–stimulated LFA-1–ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly, silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1α- and phorbol 12-myristate 13-acetate (PMA)–induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospho...
PKC-θ selectively controls the adhesion-stimulating molecule Rap1
Blood, 2008
The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. β2 integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C-θ (PKC-θ)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC-θ regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4+ T cells, clustering of ...