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.     

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-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, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.     

Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin alphaIIbbeta3

Platelet aggregation requires agonist-induced alphaIIbbeta3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered alphaIIbbeta3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates alphaIIbbeta3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to alphaIIbbeta3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or beta3 that disrupt their mutual interaction block both talin recruitment and alphaIIbbeta3 activation. However, one talin mutant (L325R) is recruited to alphaIIbbeta3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated alphaIIbbeta3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and alphaIIbbeta3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to alphaIIbbeta3 by RIAM mediates agonist-induced alphaIIbbeta3 activation, with implications for hemostasis and thrombosis.     

RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.     

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.     

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.     

Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser(756) of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.     

Dual regulation of RA-RhoGAP activity by phosphatidic acid and Rap1 during neurite outgrowth

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.     

Structural Basis for the Interaction of the Adaptor Protein Grb14 with Activated Ras

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.     

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.     

Differential Binding of Active and Inactive Integrin to Talin

Bi-directional signaling of integrins plays an important role in platelet and leukocyte function. Talin plays a key role in integrin bi-directional signaling and its binding to integrin is highly regulated. The precise regulation of the recruitment and binding of talin to integrin is still being elucidated. In particular, the recruitment of talin to integrin is controlled by the RAP-1 and RIAM/lamellipodin signaling axis and the affinity between talin and integrin is regulated by the conformation or protease cleavage of talin. However, whether the binding between integrin and talin is also regulated by integrin conformation has not been thoroughly explored before. In this work, we used biochemical binding assays to study the potential role of integrin conformational changes in integrin–talin interactions. Constitutively active integrin αIIbb3 binds markedly stronger to talin than inactive αIIbb3. Inactive αIIbb3 markedly increases its binding to talin once activated, regardless of how αIIbb3 is activated. Further, the increased binding to talin is b3 tail dependent. Our results suggest that integrin conformation is another regulatory mechanism for integrin–talin interaction.     

Structural basis for the autoinhibition of talin in regulating integrin activation

Activation of heterodimeric (alpha/beta) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin beta membrane-proximal cytoplasmic tails (beta-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin beta-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.     

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.     

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P₃; K_D ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P₃ as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P₂ on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P₃ is affected by the presence of PO₄ ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.     

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.     

The N-terminal domains of talin cooperate with the phosphotyrosine binding-like domain to activate beta1 and beta3 integrins

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.     

Structural basis for small G protein effector interaction of Ras-related protein 1 (Rap1) and adaptor protein Krev interaction trapped 1 (KRIT1)

Cerebral cavernous malformations (CCMs) affect 0.1–0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Å co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain “modules.”