Ligand-dependent activation of integrin alpha vbeta 3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.     

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.     

Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.     

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.     

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.     

Two distinct proton binding sites in the ATP synthase family

The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.     

ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.     

RGD-independent binding of integrin alpha9beta1 to the ADAM-12 and -15 disintegrin domains mediates cell-cell interaction

ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.     

Two distinct cytoplasmic regions of the beta2 integrin chain regulate RhoA function during phagocytosis

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.     

Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin alpha(IIb)beta(3)

Integrin alpha(IIb)beta(3), a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with alpha(IIb)beta(3) has been controversial. Ligand-mimetic anti-alpha(IIb)beta(3) antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in alpha(IIb) and beta(3) using human-to-mouse chimeras, which we expect to maintain functional integrity of alpha(IIb)beta(3). Here we report that these antibodies recognize several discontinuous binding sites in both the alpha(IIb) and beta(3) subunits; these binding sites are located in residues 156-162 and 229-230 of alpha(IIb) and residues 179-183 of beta(3). In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4-7, of alpha(IIb)). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.     

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.     

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.     

Two phosphorylation sites on eIF-2 alpha

Protein synthesis in mammalian cells can be regulated through phosphorylation/dephosphorylation of the alpha subunit of initiation factor 2, eIF-2. Two specific kinases have been identified that apparently phosphorylate the same site(s). Controversy exists as to whether serine-48 is a phosphorylation site in addition to serine-51. A recent publication is discussed that, in this author's view, answers the question of the phosphorylation sites. It is suggested that phosphorylation proceeds sequentially with serine-51 being the first and serine-48 the second phosphorylation site. Phosphorylation of both sites is required for inhibition of protein synthesis.     

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.