Beta2 Integrin Activation And Signaling
Vineet Gupta
Medicineuniversity Of Miami School Of Medicine
Grant 7K01DK068253-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK
Abstract: The leukocyte specific beta2 integrins are central to the biological function of these cells. Beta2 integrins mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell-mediated killing, beta2 integrins also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications, where the receptors are pathologically activated. Therefore, beta2 integrins are important therapeutic targets in inflammation, autoimmunity, and transplantation. Integrins use bi-directional signaling to regulate cellular functions. Inside-out signals activate integrins by inducing conformational changes in their extracellular ligand-binding domains. Structural basis for integrin activation is poorly understood and is a focus of intense current research. Recently described high-resolution alphaVbeta3 integrin crystal structures highlight distinctive features of the integrin dimers and provide new insights into the nature of their activation. Our aim is to develop and test specific, structure-based hypotheses for integrin activation, with a focus on beta2 integrin CD11b/CD18, by using alphaVbeta3 structure as the model for the CD11b/CD18 integrin. We will generate mutations in integrin subunits and study their effect on integrin activation and biological function using cell-based and biochemical assays. In aim 1, we will evaluate the basis of metal ion coordination at alpha-Genu, and determine the role of flexion in allosteric integrin activation. Alpha-genu is the knee structure in the alpha-subunit that lies between the thigh and the calf-1 domains. The structure serves as a highly flexible hinge, and is the site of the bend in the crystal structure that has a divalent metal ion associated with it. In aim 2, we will examine the deadbolt hypothesis and study the role of betaTD in inside-out activation of integrins. Beta tail domain (betaTD) is a novel domain that was found in beta-subunit in the integrin crystal structure. Comparison of unliganded and liganded integrin structures shows that this domain may regulate integrin activation by binding with the betaA domain and thereby acting as a deadbolt to keep integrins in an inactive form. In aim 3, we will determine the function of a extracellular juxta-membrane residue in integrin homo-oligomerization and clustering. Sequence alignment between various integrins shows that there are a number of conserved residues between the betaTD and the transmembrane region. This residue may play a part in integrin activation or clustering. Given the importance of beta2 integrins in every aspect of leukocyte function, these studies will have a profound impact on cell biology and will facilitate design of novel therapeutics to treat the many diseases resulting from pathologic modulation of integrins. The insights gained from these studies are also likely to extend to other integrins
Keywords: biological signal transduction, cell biology, integrin, posttranslational modification cell mediated cytotoxicity, cell migration, ion transport, leukocyte adhesion molecule, leukocyte oxidative burst, membrane protein, protein binding cell line, enzyme linked immunosorbent assay, flow cytometry, immunoprecipitation, site directed mutagenesis, transfection, western blotting
Project start date: 2004-08-01
Project end date: 2009-06-30
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Grants awarded to Vineet Gupta
HTS Assay For Discovery:Small Molecule Regulators (RMI)
Vineet Gupta
Massachusetts General Hospital Partners Research Management Boston, Ma 02199
Grant 1R03NS053659-01 from National Institute Of Neurological Disorders And Stroke, IRG: ZNS1
Abstract: The leukocyte specific beta2 integrins are central to the biological function of these cells. These cellular receptors mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell mediated killing. The CD11b/CD18 (alpha-M-beta2) heterodimer is the predominant beta2 integrin receptor in neutrophils and macrophages and mediates the pro-inflammatory functions in these cells. CD11b/CD18 recognizes a wide variety of ligands, including the complement fragment iC3b, fibrinogen, blood clotting factor X and CD54 (ICAM-1). Physiologic binding of CD11b/CD18 receptors to its various ligands is tightly regulated, as defects, its loss or inappropriate activation lead to severe pathological conditions and also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications. Thus, CD11b/CD18 represents an important target for small molecule drugs. Although ligand-mimetic antagonists that block CD11b/CD18 ligand binding, such as anti-CD11b/CD18 monoclonal antibodies (mAbs), have been shown to be effective in certain diseases in animals, small molecule regulators, especially allosteric regulators, are pharmacologically more desirable for human therapeutic applications. There is an inadequate array of selective and potent small molecule beta2 integrin modulators available to the public, and there are no CD11b/CD18 selective small molecule antagonists, especially allosteric inhibitors, currently available. Additionally, no CD11b/CD18 specific agonists are available at this time. A number of established functional assays, including some developed in our laboratory, can be modified and adapted for screening of small molecule regulators of CD11b/CD18. The major goals of our current proposal are to optimize and automate these available assays for a high throughput screening (HTS) environment and use them in a pilot screen to discover small molecule effectors of CD11b/CD18 (Aim 1). Furthermore, we have recently discovered a novel allosteric regulatory site, the beta tail Domain (3TD), in integrins. In this proposal, we would also like to develop HTS assays for identifying small molecules that bind at this site and thus, allosterically regulate integrin activation (Aim 2). We believe that unique molecules discovered in our screen will not only be useful as therapeutic leads against CD11b/CD18, but may also serve as novel probes for structural and mechanistic studies and as integrin conformation sensors. Such studies will undoubtedly shed new light into the mechanism of integrin activation as well. Given the central role of beta2 integrins in regulating leukocyte function, we hope that this research will provide important new tools and further guide future experimentation.
Keywords: drug discovery /isolation, drug screening /evaluation, genetic regulation, high throughput technology, inhibitor /antagonist, integrin, receptor expression, small molecule, technology /technique development, biotechnology, flow cytometry, monoclonal antibody
Project start date: 2005-09-30
Project end date: 2007-08-31
1R03NS053659-01 (2005): $87500
Beta2 Integrin Activation And Signaling
Vineet Gupta
Medicineuniversity Of Miami School Of Medicine
Grant 7K01DK068253-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK
Abstract: The leukocyte specific beta2 integrins are central to the biological function of these cells. Beta2 integrins mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell-mediated killing, beta2 integrins also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications, where the receptors are pathologically activated. Therefore, beta2 integrins are important therapeutic targets in inflammation, autoimmunity, and transplantation. Integrins use bi-directional signaling to regulate cellular functions. Inside-out signals activate integrins by inducing conformational changes in their extracellular ligand-binding domains. Structural basis for integrin activation is poorly understood and is a focus of intense current research. Recently described high-resolution alphaVbeta3 integrin crystal structures highlight distinctive features of the integrin dimers and provide new insights into the nature of their activation. Our aim is to develop and test specific, structure-based hypotheses for integrin activation, with a focus on beta2 integrin CD11b/CD18, by using alphaVbeta3 structure as the model for the CD11b/CD18 integrin. We will generate mutations in integrin subunits and study their effect on integrin activation and biological function using cell-based and biochemical assays. In aim 1, we will evaluate the basis of metal ion coordination at alpha-Genu, and determine the role of flexion in allosteric integrin activation. Alpha-genu is the knee structure in the alpha-subunit that lies between the thigh and the calf-1 domains. The structure serves as a highly flexible hinge, and is the site of the bend in the crystal structure that has a divalent metal ion associated with it. In aim 2, we will examine the deadbolt hypothesis and study the role of betaTD in inside-out activation of integrins. Beta tail domain (betaTD) is a novel domain that was found in beta-subunit in the integrin crystal structure. Comparison of unliganded and liganded integrin structures shows that this domain may regulate integrin activation by binding with the betaA domain and thereby acting as a deadbolt to keep integrins in an inactive form. In aim 3, we will determine the function of a extracellular juxta-membrane residue in integrin homo-oligomerization and clustering. Sequence alignment between various integrins shows that there are a number of conserved residues between the betaTD and the transmembrane region. This residue may play a part in integrin activation or clustering. Given the importance of beta2 integrins in every aspect of leukocyte function, these studies will have a profound impact on cell biology and will facilitate design of novel therapeutics to treat the many diseases resulting from pathologic modulation of integrins. The insights gained from these studies are also likely to extend to other integrins
Keywords: biological signal transduction, cell biology, integrin, posttranslational modification cell mediated cytotoxicity, cell migration, ion transport, leukocyte adhesion molecule, leukocyte oxidative burst, membrane protein, protein binding cell line, enzyme linked immunosorbent assay, flow cytometry, immunoprecipitation, site directed mutagenesis, transfection, western blotting
Project start date: 2004-08-01
Project end date: 2009-06-30
5K01DK068253-04 (2007): $135540
5K01DK068253-03 (2006): $131490
5K01DK068253-02 (2005): $130680
1K01DK068253-01 (2004): $129870
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