The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.

To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.     

Phosphorylation of beta3 integrin controls ligand binding strength.

The cytoplasmic domain of beta(3) integrin contains tyrosines at positions 747 and 759 in domains that have been implicated in regulation of alpha(v)beta(3) function and that serve as potential substrates for Src family kinases. The phosphorylation level of beta(3) integrin was modulated using a temperature-sensitive v-Src kinase. Increased beta(3) phosphorylation abolished alpha(v)beta(3)- but not alpha(5)beta(1)-mediated adhesion to fibronectin. alpha(v)beta(3)-Mediated cell adhesion was restored by the expression of beta(3) containing Y747F or Y759F mutations but not by wild type beta(3) integrin. Thus, phosphorylation of the cytoplasmic domain of beta(3) is a negative regulator of alpha(v)beta(3)-fibronectin binding strength.     

Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2

Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).     

M-CSF induces the stable interaction of cFms with alphaVbeta3 integrin in osteoclasts

The macrophage colony stimulating factor receptor (cFms) and alpha(V)beta(3) integrin are both abundantly expressed and play critical roles in the differentiation, survival and migration of osteoclasts. We have previously demonstrated that cross-talk between cFms- and alpha(V)beta(3)-mediated signaling pathways regulated the cytoskeletal organization required for osteoclast migration. To investigate the nature of interaction between the two receptors, we sequentially used anion-exchange chromatography and immunoprecipitation to purify alpha(V)beta(3)-associated protein complexes. We have demonstrated that cFms stably associated with alpha(V)beta(3) in osteoclasts during adhesion, and that the association was induced by macrophage colony stimulating factor (M-CSF) stimulation. However, the kinetics of association of alpha(V)beta(3) and cFms did not correlate with the kinetics of tyrosine phosphorylation of cFms. Instead, maximally observed alpha(V)beta(3)/cFms association was after the peak of cFms tyrosine phosphorylation and correlated inversely with the total amount of cFms remaining. Furthermore, the complex containing cFms and alpha(V)beta(3) also contained a number of other signaling molecules including Pyk2, p130(Cas) and c-Cbl, known downstream regulators of the integrin-mediated signaling pathways in osteoclasts. In the presence of M-CSF, co-localization of alpha(V)beta(3) integrin and cFms was identified in the podosomal actin ring of the osteoclast during adhesion on glass. Interestingly, co-localization of both receptors was not found in the sealing zone, but in punctate structures associated with adhesion- or transcytosis-like structures in osteoclasts on bone. Taken together, we suggest that the association of alpha(V)beta(3) and cFms could be the result of signaling following tyrosine phosphorylation of cFms. The recruitment of cFms to alpha(V)beta(3) integrin may be an integral part of a larger signaling complex via which both of adhesion- and growth factor receptors coordinately regulate osteoclast adhesion, motility and membrane trafficking.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.     

Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).     

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.     

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.     

Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.     

Activation of Rac and tyrosine phosphorylation of cytokine receptors induced by cross-linking of integrin alpha4beta1 and cell adhesion in hematopoietic cells

Adhesion of hematopoietic cells, mainly through alpha4beta1 and alpha5beta1 integrins, to the bone marrow microenvironment may play important roles in regulation of hematopoiesis. However, the mechanisms for signaling, outside-in signaling, have largely remained to be established. We demonstrate here that cross-linking of alpha4beta1 by anti-alpha4 antibody induces tyrosine phosphorylation of Pyk2, Shc, and Cbl as well as binding of the adaptor protein CrkL with Cbl in a murine hematopoietic cell line, 32D/EpoR-Wt. Furthermore, cross-linking of alpha4beta1 induced activation of the Rho family small GTPase Rac, which was enhanced by induced overexpression of CrkL and was inhibited by the phosphatidylinositol 3(')-kinase (PI3K) inhibitor LY294002. In addition, adhesion of 32D/EpoR-Wt cells to immobilized H-296, a recombinant fibronectin peptide specific for alpha4beta1, induced tyrosine phosphorylation of Jak2, the erythropoietin receptor (EpoR), and the IL-3 receptor beta subunit as well as Pyk2, Shc, and Cbl. Tyrosine phosphorylation of Jak2 and EpoR was also induced in a human leukemic cell line, UT-7, by adhesion to immobilized H-296. However, adhesion of 32D/EpoR-PM4 cells, expressing the W282R mutant EpoR defective in coupling with Jak2, to immobilized H-296 failed to induce tyrosine phosphorylation of the mutant EpoR. These results implicate CrkL in PI3K-dependent activation of Rac by outside-in signaling from alpha4beta1 and suggest that adhesion through alpha4beta1 further activates cytokine receptor-associated Jak2 to induce phosphorylation of these receptors.     

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

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.     

Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling

Outside-in signaling mediated by the integrin alpha(IIb)beta(3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta(3). To identify proteins that bind directly to phosphorylated beta(3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta(3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated beta(3). To determine the involvement of Shc in outside-in alpha(IIb)beta(3) signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated beta(3) in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated beta(3) peptides was direct, demonstrating Shc recognition motifs on phospho-beta(3). Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) beta(3), which are defective in outside-in alpha(IIb)beta(3) signaling. Thus, Shc is the primary downstream signaling partner of beta(3) in its tyrosine phosphorylation outside-in signaling pathway.     

Compartmentalization of integrin alpha6beta4 signaling in lipid rafts

Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.     

A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function.

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Kinetic regulation of beta 3 integrin tyrosine phosphorylation

Tyrosine phosphorylation of beta(3) integrins is a permissive stage in the activation of alpha(IIb)beta(3) and alpha(v)beta(3) in platelets and leukocytes, respectively. In this study we demonstrated direct phosphorylation of beta(3) integrins as a result of interaction with soluble monomeric ligand, and we characterized the differential kinetics of beta(3) phosphorylation as a consequence of alpha subunit pairing. We found that beta(3) phosphorylation is initiated by RGD peptide binding in a dose-dependent and saturable fashion with alpha(IIb)beta(3) becoming phosphorylated and dephosphorylated more rapidly than alpha(v)beta(3). Site mapping of phosphate incorporation reveals significant phosphorylation at Tyr-747 in both beta(3) integrin species with incorporation at Tyr-759 found at significant levels only in alpha(IIb)beta(3). Mutation of cytoplasmic beta(3) tyrosine residues in a transfection model prevents cell adhesion via these integrins. These data demonstrate that recognition of ligand is sufficient to induce beta(3) tyrosine phosphorylation and suggests that this event is regulated by the alpha subunit pairing of beta(3).     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.