Binding properties and stability of the Ras-association domain of Rap1-GTP interacting adapter molecule (RIAM)

The Rap1-GTP interacting adapter protein (RIAM) is an important protein in Rap1-mediated integrin activation. By binding to both Rap1 GTPase and talin, RIAM recruits talin to the cell membrane, thus facilitating talin-dependent integrin activation. In this article, we studied the role of the RIAM Ras-association (RA) and pleckstrin-homology (PH) domains in the interaction with Rap1. We found that the RA domain was sufficient for GTP-dependent interaction with Rap1B, and the addition of the PH domain did not change the binding affinity. We also detected GTP-independent interaction of Rap1B with the N-terminus of RIAM. In addition, we found that the PH domain stabilized the RA domain both in vitro and in cells.     

RIAM links the ADAP/SKAP-55 signaling module to Rap1, facilitating T-cell-receptor-mediated integrin activation

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.     

Rap1-GTP-interacting adaptor molecule (RIAM) protein controls invasion and growth of melanoma cells

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces β1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.     

Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3

BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.     

Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity

Epithelial tight junctions form a selectively permeable barrier to ions and small molecules. Junctional adhesion molecule 1 (JAM1/JAM-A/F11R) is a tight junction-associated transmembrane protein that has been shown to participate in the regulation of epithelial barrier function. In a recent study, we presented evidence suggesting that JAM1 homodimer formation is critical for epithelial barrier function (Mandell, K. J., McCall, I. C., and Parkos, C. A. (2004) J. Biol. Chem. 279, 16254-16262). Here we have used small interfering RNA to investigate the effect of the loss of JAM1 expression on epithelial cell function. Consistent with our previous study, knockdown of JAM1 was observed to increase paracellular permeability in epithelial monolayers. Interestingly, knockdown of JAM1 also produced dramatic changes in cell morphology, and a similar effect was observed with expression of a JAM1 mutant lacking the putative homodimer interface. Further studies revealed that JAM1 knockdown decreased cell-matrix adhesion and spreading on matrix proteins that are ligands of beta1 integrins. These changes were characterized by a decrease in beta1 integrin protein levels and loss of beta1 integrin staining at the cell surface. Immunolabeling of cells for the small GTPase Rap1, a known activator of beta1 integrins, revealed colocalization of Rap1 with JAM1 at intercellular junctions, and knockdown of JAM1 resulted in decreased Rap1 activity. Lastly, knockdown of Rap1b resulted in diminished beta1 integrin expression and altered cell morphology analogous to that observed with knockdown of JAM1. Together, these results suggest that JAM1 regulates epithelial cell morphology and beta1 integrin expression by modulating activity of the small GTPase Rap1.     

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P₂) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P₂.     

RIAM Activates Integrins by Linking Talin to Ras GTPase Membrane-targeting Sequences

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1). Each integrin comprises a type I transmembrane α and β subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin β cytoplasmic domains and the binding of talin to the integrin β tail initiates integrin activation (2–4). A small, PTB-like domain of talin mediates activation via a two-site interaction with integrin β tails (5), and this PTB domain is functionally masked in the intact talin molecule (6). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs)³ enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9, 10). For example, expression of constitutively active Rap1 activates integrin αMβ2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11–13). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14). In platelets, Rap1 is rapidly activated by platelet agonists (15, 16). A knock-out of Rap1B (17) or of the Rap1GEF, RasGRP2 (18), resulted in impairment of αIIbβ3-dependent platelet aggregation, highlighting the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1 GTPases play important roles in the activation of several integrins in multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation (19–21). Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins (20). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features of the MRL protein family. In Jurkat cells, RIAM overexpression induces β1 and β2 integrin-mediated cell adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion (20), indicating RIAM is a downstream regulator of Rap1-dependent signaling. RIAM regulates actin dynamics as RIAM expression induces cell spreading; conversely, its depletion reduces cellular F-actin content (20). Whereas RIAM is greatly enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in fibroblasts and other somatic cells (22).Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in Chinese hamster ovary cells expressing a prototype activable integrin, platelet αIIbβ3. We found that Rap1 induced formation of an “integrin activation complex” containing RIAM and talin (23). Here, we have established the mechanism whereby Ras GTPases cooperate with MRL family proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma membrane.     

Rap1 translates chemokine signals to integrin activation,cell polarization,and motility across vascular endothelium under flow

Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.     

The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes

Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365-1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756-41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and alpha(4) integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.     

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(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 GTPase-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 GTP-bound 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.     

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

AF6 negatively regulates Rap1-induced cell adhesion

AF6 is involved in the connection of membrane-associated proteins to the actin cytoskeleton. It binds to Ras-like small GTPases and is suggested to be an effector of both Ras and Rap. Here we show that knockdown of AF6 in T cells by RNA interference enhanced Rap1-induced integrin-mediated cell adhesion, whereas overexpression of AF6 had the opposite effect. Interestingly, AF6-induced inhibition of cell adhesion correlated with an increase in RapGTP levels. Like AF6, protein KIAA1849 contains a Ras association domain and interacted with Rap1. However, KIAA1849 did not inhibit Rap1-induced cell adhesion. We concluded that AF6 is a negative regulator of Rap-induced cell adhesion. We proposed that AF6 inhibits Rap-mediated cell adhesion by sequestering RapGTP in an unproductive complex and thus prevents the interaction of Rap1 not only with effectors that mediate adhesion but also with Rap GTPase-activating proteins. Thus, AF6 may buffer RapGTP in resting T cells and maintain them in a non-adherent state.     

The GTPase Rap1 regulates phorbol 12-myristate 13-acetate-stimulated but not ligand-induced beta 1 integrin-dependent leukocyte adhesion

Leukocyte migration from bloodstream to tissue requires rapid, coordinated regulation of integrin-dependent adhesion and de-adhesion. In a previous study we demonstrated that inhibition of protein geranylgeranylation inhibited phorbol ester-stimulated avidity modulation of beta(1) integrin in several leukocyte cell lines. Both RhoA and Rap1 require post-translational modification by geranylgeranylation for full function. In this report we identify Rap1, not RhoA, as a critical geranylgeranylated protein mediating phorbol ester-stimulated beta(1) and beta(2) integrin-dependent adhesion of Jurkat cells. Overexpression of the Rap1-specific GTPase-activating protein, SPA-1, or inactivated form of Rap1 (N17Rap1) blocked phorbol ester-stimulated adhesion of Jurkat cells to fibronectin (alpha(4)beta(1)) and ICAM-1 (alpha(L)beta(2)). With high concentrations of fibronectin as ligand, Jurkat cells adhered spontaneously without phorbol ester stimulation. Unlike the phorbol ester-stimulated adhesion, adhesion induced by high density ligand was not dependent upon Rap1 activation or actin cytoskeleton reorganization. Thus, the "inside-out" adhesion signal induced by phorbol ester and the "outside-in" signal induced by high density ligand involve different pathways.     

The Ras-related Protein,Rap1A,Mediates Thrombin-stimulated,Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β₁ integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β₁ integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β₁ integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.     

Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl^−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl^−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein–protein interactions.     

Recent advances in the understanding of the molecular mechanisms regulating platelet integrin αIIbβ3 activation

Integrins are allosteric cell adhesion receptors that cycle from a low to a high affinity ligand binding state, a complex process of receptor activation that is of particular importance in blood cells such as platelets or leukocytes. Here we highlight recent progress in the understanding of the molecular pathways that regulate integrin activation in platelets and leukocytes, with a special focus on the structural changes in platelet integrin αIIbβ3 brought about by key intracellular proteins, namely talin and kindlins, that are of crucial importance in the regulation of integrin function. Evidence that the small GTPase Rap1 and its guanine exchange factor CalDAG-GEF1, together with RIAM, a Rap1GTP adaptor protein, promote the interaction of talin with the integrin β subunit, has greatly contributed to fill the gap in our understanding of the signaling pathway from G-coupled agonist receptors and their phospholipase C-dependant second messengers, to integrin activation. Studies of patients with the rare blood cell disorder LAD-III have contributed to the identification of kindlins as new co-regulators of the talin-dependent integrin activation process in platelets and leukocytes, underlining the relevance for the in-depth investigation of patients with rare genetic blood cell disorders.     

Rap1 controls activation of the αMβ2 integrin in a talin‐dependent manner

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin α_Mβ₂ in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with α_Mβ₂, we show that Rap1 acts on the β₂ and not the α_M chain, and that residues 732–761 of the β₂ subunit are essential for Rap1‐induced RBC binding. Activation of α_Mβ₂ by Rap1 was dependent on W747 and F754 in the β₂ tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of α_Mβ₂ in macrophages shares both common and distinct features from Rap1 activation of α_IIbβ₃ expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc.     

Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion.