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Horst Kessler - One of the best experts on this subject based on the ideXlab platform.

  • multimeric cyclic rgd peptides as potential tools for tumor targeting solid phase peptide synthesis and chemoselective oxime ligation
    Chemistry: A European Journal, 2003
    Co-Authors: Georgette Thumshirn, Ulrich Hersel, Simon L Goodman, Horst Kessler
    Abstract:

    The αvβ3 Integrin Receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this Receptor may provide information about the Receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced Integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging αvβ3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.

  • n methylated cyclic rgd peptides as highly active and selective alpha v beta 3 Integrin antagonists
    Journal of Medicinal Chemistry, 1999
    Co-Authors: Michael A Dechantsreiter, Simon L Goodman, Eckart Planker, Barbara Matha, Elisabeth Lohof, Gunter Holzemann, Alfred Jonczyk, Horst Kessler
    Abstract:

    The alpha(V)beta(3) Integrin Receptor plays an important role in human tumor metastasis and tumor-induced angiogenesis. The in vivo inhibition of this Receptor by antibodies or by cyclic peptides containing the RGD sequence may in the future be used to selectively suppress these diseases. Here we investigate the influence of N-methylation of the active and selective alpha(V)beta(3) antagonist cyclo(RGDfV) (L1) on biological activity. Cyclo(RGDf-N(Me)V-) (P5) was found to be even more active than L1 and is one of the most active and selective compounds in inhibiting vitronectin binding to the alpha(V)beta(3) Integrin. Its high-resolution, three-dimensional structure in water was determined by NMR techniques, distance geometry calculations, and molecular dynamics calculations, providing more insight into the structure-activity relationship.

David D. Schlaepfer - One of the best experts on this subject based on the ideXlab platform.

  • Focal adhesion kinase signaling in unexpected places.
    Current opinion in cell biology, 2017
    Co-Authors: Elizabeth G. Kleinschmidt, David D. Schlaepfer
    Abstract:

    Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase first identified at extracellular matrix and Integrin Receptor cell adhesion sites and is a key regulator of cell movement. FAK is activated by a variety of stimuli. Herein, we discuss advances in conformational-associated FAK activation and dimerization mechanisms. Additionally, new roles have emerged for FAK signaling at cell adhesions, adherens junctions, endosomes, and the nucleus. In light of these new findings, we review how FAK activation at these sites is connected to the regulation of Integrin recycling-activation, vascular permeability, cell survival, and transcriptional regulation, respectively. Studies uncovering FAK signaling connections in unexpected places within cells have yielded important new regulatory insights in cell biology.

  • required role of focal adhesion kinase fak for Integrin stimulated cell migration
    Journal of Cell Science, 1999
    Co-Authors: David J Sieg, Christof R Hauck, David D. Schlaepfer
    Abstract:

    FAK localizes to sites of transmembrane Integrin Receptor clustering and facilitates intracellular signaling events. FAK-null (FAK-) fibroblasts exhibit a rounded morphology, defects in cell migration, and an elevated number of cell-substratum contact sites. Here we show that stable re-expression of epitope-tagged FAK reversed the morphological defects of the FAK- cells through the dynamic regulation of actin structures and focal contact sites in fibronectin (FN) stimulated cells. FAK re-expressing fibroblasts (clones DA2 and DP3) exhibit a characteristic fibrillar shape and display indistinguishable FN Receptor-stimulated migration properties compared to normal fibroblasts. Expression of various FAK mutants in the FAK- cells showed that FAK kinase activity, the Tyr-397/SH2 domain binding site, and the first proline-rich SH3 binding region in the FAK C-terminal domain were individually needed to promote full FAK-mediated FAK- cell migration to FN whereas direct paxillin binding to FAK was not required. Expression of the FAK Phe-397 mutant did not promote FAK- cell migration and overexpression of p50(csk) in DA2 cells inhibited migration to FN suggesting that Src-family PTKs play important roles in FAK-mediated motility events. Expression of the FAK C-terminal domain, FRNK, promoted FAK dephosphorylation at Tyr-397 and potently blocked FAK-mediated cell migration. This dominant-negative effect of FRNK was reversed by a point mutation (Leu-1034 to Ser) which prevented FRNK localization to focal contact sites. Our results show that FAK functions as a key regulator of fibronectin Receptor stimulated cell migration events through the recruitment of both SH2 and SH3 domain-containing signaling proteins to sites of Integrin Receptor clustering.

  • signaling through focal adhesion kinase
    Progress in Biophysics & Molecular Biology, 1999
    Co-Authors: David D. Schlaepfer, Christof R Hauck, David J Sieg
    Abstract:

    Integrin Receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking Integrin Receptors to intracellular signaling pathways. FAK associates with several diAerent signaling proteins such as Src-family PTKs, p130 Cas , Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of Integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAKmediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by Integrin Receptor-mediated events and also non-Integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of Integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK ˇ ) and how these cells exhibit deficits

  • signaling through focal adhesion kinase
    Progress in Biophysics & Molecular Biology, 1999
    Co-Authors: David D. Schlaepfer, Christof R Hauck, David J Sieg
    Abstract:

    Integrin Receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking Integrin Receptors to intracellular signaling pathways. FAK associates with several diAerent signaling proteins such as Src-family PTKs, p130 Cas , Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of Integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAKmediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by Integrin Receptor-mediated events and also non-Integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of Integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK ˇ ) and how these cells exhibit deficits

J Cochran - One of the best experts on this subject based on the ideXlab platform.

  • preliminary evaluation of 177lu labeled knottin peptides for Integrin Receptor targeted radionuclide therapy
    European Journal of Nuclear Medicine and Molecular Imaging, 2011
    Co-Authors: J Cochran, Lei Jiang, Zheng Miao, Richard H Kimura, Hongguang Liu, Cathy S Culter, Ande Bao, Zhen Cheng
    Abstract:

    Purpose Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind Integrin Receptors with high affinity and specificity. These Receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, 177Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts.

  • engineered cystine knot peptides that bind αvβ3 Integrin with antibody like affinities
    Journal of Molecular Biology, 2009
    Co-Authors: Adam P Silverman, Aron M Levin, Jennifer L Lahti, J Cochran
    Abstract:

    Abstract The α v β 3 Integrin Receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to α v β 3 Integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp Integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized α v β 3 Integrin Receptor. Select Integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various Integrin Receptors. These studies showed that the engineered AgRP peptides bound to cells expressing α v β 3 Integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to α v β 3 Integrins and had only minimal or no binding to α v β 5 , α 5 β 1 , and α iib β 3 Integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for α v β 3 and other Integrins. Next, to evaluate whether the other three loops of AgRP could modulate Integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity Integrin-binding variants. Screening of these loop-randomized libraries against α v β 3 Integrins resulted in peptides that retained high affinities for α v β 3 and had increased specificities for α v β 3 over α iib β 3 Integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.

Cay M. Kielty - One of the best experts on this subject based on the ideXlab platform.

  • Fibrillin-1 microfibril deposition is dependent on fibronectin assembly.
    Journal of cell science, 2008
    Co-Authors: Rachel Kinsey, Matthew R Williamson, Shazia Chaudhry, Amanda Mcgovern, Seiichiro Takahashi, Kieran T Mellody, C Adrian Shuttleworth, Cay M. Kielty
    Abstract:

    Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As Integrin Receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of beta1-Integrin-Receptor-null fibroblasts, blockage of cell surface Integrin Receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of beta1 Integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectin(RGE/RGE) mutant mouse fibroblast cultures, which do not engage alpha5beta1 Integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

  • Fibrillin-1 microfibril deposition is dependent on fibronectin assembly
    Journal of Cell Science, 2008
    Co-Authors: Rachel Kinsey, Matthew R Williamson, Amanda Mcgovern, Seiichiro Takahashi, Kieran T Mellody, Shazia S Chaudhry, C Adrian Shuttleworth, Cay M. Kielty
    Abstract:

    Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As Integrin Receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of β1-Integrin-Receptor-null fibroblasts, blockage of cell surface Integrin Receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of β1 Integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectin RGE/RGE mutant mouse fibroblast cultures, which do not engage α5β1 Integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

Paula J Mckeownlongo - One of the best experts on this subject based on the ideXlab platform.

  • the Receptor for urokinase type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts
    Journal of Biological Chemistry, 2004
    Co-Authors: Elizabeth Monaghan, Volodia D Gueorguiev, Cynthia E Wilkinsport, Paula J Mckeownlongo
    Abstract:

    Abstract Previous studies have indicated that the Receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with Integrin Receptors thereby modulating Integrin activity. In the present study, the role of uPAR in the regulation of α5β1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the β1 Integrin Receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in α5β1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the β1 Integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the α5β1 Integrin Receptor and suggest that changes in Integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and Integrin Receptors synergistically regulate the levels of fibronectin in the extracellular matrix.

  • αvβ5 Integrin Receptor mediated endocytosis of vitronectin is protein kinase c dependent
    Journal of Biological Chemistry, 1995
    Co-Authors: T S Panetti, Sarah A Wilcox, Carol Horzempa, Paula J Mckeownlongo
    Abstract:

    Abstract Previous studies have demonstrated that the αvβ5 Integrin Receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J.(1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J.(1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the β5 Integrin or exogenous heparin, suggesting that both Integrin Receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the αvβ5 Integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-αvβ5 antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the αvβ5 Integrin following activation of protein kinase C.

  • the alpha v beta 5 Integrin Receptor regulates Receptor mediated endocytosis of vitronectin
    Journal of Biological Chemistry, 1993
    Co-Authors: T S Panetti, Paula J Mckeownlongo
    Abstract:

    Abstract Vitronectin is an adhesive glycoprotein that binds to the extracellular matrix and interacts with Integrin Receptors on the surface of adherent cells. Previous studies have demonstrated that the conformationally altered, heparin binding form of vitronectin is removed from the matrix by Receptor-mediated endocytosis and degraded through a lysosomal pathway (Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993). The present studies were undertaken to determine the role of cell surface Integrins in the endocytosis and degradation of vitronectin. RGDS peptides, used to disrupt the binding of vitronectin to cell surface Integrins, inhibited degradation of vitronectin but had no effect on the binding of vitronectin to the cell layer. Localization of vitronectin in the cell layer by indirect immunofluorescence indicated that the RGDS peptides inhibited degradation by preventing the internalization of vitronectin by the cells. To determine which vitronectin Receptor was involved in mediating the endocytosis, vitronectin degradation was measured in the presence of monoclonal antibodies. Antibodies against the alpha v beta 5 but not the alpha v beta 3 Integrin inhibited degradation of vitronectin by 80%. This study demonstrates a new role for Integrins in regulating internalization and degradation of molecules from the extracellular matrix.