David A Holowka
Cornell University Ithaca
Project start date: 1985-08-01
Project end date: 2014-12-31
Sponsored Links Excellgen http://Excellgen.com
ANTIGEN BINDING, CROSSLINKING & TRANSMEMBRANE SIGNALING
David A Holowka, Senior Scientist
Chemistry And Chemical Biologycornell University Ithaca
office Of Sponsored Programs
ithaca, Ny 148502820
Grant 5R01AI022449-15 from National Institute Of Allergy And Infectious Diseases IRG: ZRG2
Abstract: The proposed studies will advance elucidation of the features of antigen-mediated crosslinking of lgE bound to its high affinity receptor, Fc epsilonRI, on mast cells that are critical for initiating a cascade of signaling events leading to the release of key modiators in allergic responses. The studies will build on strong foundation of fluorescence and other methodologies and concepts that are derived from previous work on this well-characterized model immune cell system. We have established that a small, symmetrical bivalent ligand stimulates a cellular response depending on whether this ligand forms small cyclic complexes or linear chains with lgE bound to Fc epsilon RI , providing evidence that structural restrictions between crosslinked lgE-Fc epsilon RI can limit cell activation. This hypothesis will be tested in Specific Aim 1 with a new generation of rigid bivalent ligands made with double-stranded DNA, together with a specially prepared bi- specific lgE. The structural arrangement of crosslinked lgE-Fc epsilon Rl beta and gamma subunits by Lyn, and the binding and activation of the tyrosine kinase Syk. Binding of trivalent and multivalent antigens will also be analyzed in conjunction with their functional activities. Specific Aim 2 will investigate the functional importance of specialized plasma membrane domains that are being characterized in this laboratory. Crosslinking of lgE-Fc epsilon Ri causes association with detergent-resistant membrane domains that contain abundant Lyn tyrosine kinase activity, and our recent results indicate that these interactions are important for initiating Fc epsilon Rl mediated signal transduction. These domain-Fc epsilon Rl interactions, their regulation by the mirofilament cytoskeleton, and the consequences of these on early and late signaling events will be investigated in biochemical preparations and intact cells. Because of the central role of ligand binding and receptor aggregation in immune cell activation and the accumulating evidence that signaling molecules localized to membrane domains are integrally involved, the proposed investigation should lead to new concepts and targets for therapeutic intervention
Keywords: antibody receptor, biological signal transduction, crosslink, hypersensitivity, immunoglobulin E, receptor binding DNA, cell membrane, mast cell, membrane activity, membrane lipid, phosphorylation, protein tyrosine kinase flow cytometry, tissue /cell culture
Project start date: 1985-08-01
Project end date: 2002-06-30
5R01AI022449-15 (2001): $229903
5R01AI022449-14 (2000): $223207
5R01AI022449-13 (1999): $216705
5R01AI022449-12 (1998): $210393
Antigen Binding, Crosslinking And Signaling
David A Holowka, Senior Scientist
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 5R01AI022449-20 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: The proposed research addresses key unanswered questions about the structures and events by which antigen-activated immunoreceptors initiate cellular signaling leading to diverse responses. The high affinity receptor for lgE (FcepsilonRI) on RBL-2H3 mast cells serves as a valuable model system, and new experimental thrusts will build on previous extensive studies. Specific Aim #1 extends experiments with structurally defined ligands to extract critical features of ligand architecture and cross-linking mechanism that facilitate or inhibit signaling responses. Specific Aim #2 continues efforts to understand the molecular basis by which lipid rafts participate in FcepsilonRI signal initiation, and to achieve functional reconstitution of this receptor in model membranes from purified, defined components. Specific Aim #3 proposes real-time imaging studies to elucidate the process by which FcepsilonRI becomes segregated from particular lipid raft components, and investigate how plasma membrane locations for stimulated exocytosis are determined. Specific Aim #4 proposes experiments to understand the mechanisms by which Rho family proteins and other low molecular weight GTPases participate in early FcepsilonRI signaling, as revealed by previous studies with mutant RBL-B6A4C1 mast cells. These investigations integrate diverse physical, chemical, and biological approaches to provide novel insights into the structure and molecular mechanisms of IgE-FcepsilonRI signaling, and they will advance our molecular understanding of the cell biology of this important immune cell prototype.
Keywords: antibody receptor, antigen receptor, biological signal transduction, cellular immunity, crosslink, immunoglobulin E, receptor binding, B cell receptor, T cell receptor, exocytosis, granulocyte, guanosinetriphosphatase, mast cell, phosphorylation, protein tyrosine kinase, CHO cell, flow cytometry, tissue /cell culture, western blotting
Project start date: 1985-08-01
Project end date: 2008-04-30
5R01AI022449-20 (2007): $245752
5R01AI022449-19 (2006): $252499
5R01AI022449-18 (2005): $259387
5R01AI022449-17 (2004): $260169
ANTIGEN BINDING, CROSSLINKING & TRANSMEMBRANE SIGNALING
David A Holowka, Senior Scientist
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 5R01AI022449-07 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: A process of fundamental importance in immunology is the binding of antigen to cell surface-associated immunoglobulin that leads to transmembrane signaling in specialized cells. Antigen-mediated crosslinking of immunoglobulin E-receptor complexes on mast cells and basophils leads to degranulation in the allergic response, while crosslinking of surface immunoglobulin on B lymphocytes by certain antigens is a primary singal for proliferation and differentiation into antibody secreting cells. The critical molecular features of the crosslinking events that lead to a bioloogical response in either of these systems are not understood, and the proposed studies are aimed at elucidating these features. In previous studies on the IgE-receptor system, fluorescence methods have been developed that have allowed the kinetics of binding and crosslinking by model bivalent antigens to e determined. These studies have revealsed that some bivalent antigens can crosslink IgE-receptor complexes very efficiently on the cell surface, yet trigger only a small cellulr degranulation response. Properties such as rate of crosslinking, crosslink lifetime, and rate of dissociation for these antigens will be compared with those of other bivalent antigens that trighger a much stronger biological response. By this means the relationship between these kinetic properties, activation, and inactivation (densensitization) signals will be investigated. Structural factors also play a role in determining the efficacy of crosslinking, and these will be studied using model bivalent antigens of different lengths. Distances between IgE-receptor complexes crosslinked by long, rigid bivalent antigens that trigger degranulation will be mapped by electron microscopic and resonance energy transfer methods. The methodologies developed on the IgE-receptor system will be extended to studies of model antigen binding and crosslinking of dansyl-specific surface immunoglobulin on B cells and hybridoma cell lines. These studies will determine whether the features of crosslinking that are critical for signal tranduction by IgE- receptor complexes on mast cells are also important for transmembrane signaling by surface immunoglobulin on B cells.
Keywords: biological signal transduction, crosslink, lymphocyte proliferation, receptor binding, B lymphocyte, T cell receptor, T lymphocyte, antigen receptor, calcium flux, cellular immunity, chemical binding, chemical reaction, hapten, hybridoma, immunoglobulin E, mast cell, membrane activity, membrane protein, protein kinase C, avidin, electron microscopy, monoclonal antibody, tissue /cell culture
Project start date: 1985-08-01
Project end date: 1993-02-28
5R01AI022449-07 (1992): $149202
5R01AI022449-09 (1994): $184295
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to David A Holowka
ANTIGEN BINDING, CROSSLINKING And TRANSMEMBRANE SIGNALING
David A Holowka, Senior Scientist
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 2R01AI022449-11A2 from National Institute Of Allergy And Infectious Diseases IRG: ZRG2
Abstract: The proposed studies will advance elucidation of the features of antigen-mediated crosslinking of lgE bound to its high affinity receptor, Fc epsilonRI, on mast cells that are critical for initiating a cascade of signaling events leading to the release of key modiators in allergic responses. The studies will build on strong foundation of fluorescence and other methodologies and concepts that are derived from previous work on this well-characterized model immune cell system. We have established that a small, symmetrical bivalent ligand stimulates a cellular response depending on whether this ligand forms small cyclic complexes or linear chains with lgE bound to Fc epsilon RI , providing evidence that structural restrictions between crosslinked lgE-Fc epsilon RI can limit cell activation. This hypothesis will be tested in Specific Aim 1 with a new generation of rigid bivalent ligands made with double-stranded DNA, together with a specially prepared bi- specific lgE. The structural arrangement of crosslinked lgE-Fc epsilon Rl beta and gamma subunits by Lyn, and the binding and activation of the tyrosine kinase Syk. Binding of trivalent and multivalent antigens will also be analyzed in conjunction with their functional activities. Specific Aim 2 will investigate the functional importance of specialized plasma membrane domains that are being characterized in this laboratory. Crosslinking of lgE-Fc epsilon Ri causes association with detergent-resistant membrane domains that contain abundant Lyn tyrosine kinase activity, and our recent results indicate that these interactions are important for initiating Fc epsilon Rl mediated signal transduction. These domain-Fc epsilon Rl interactions, their regulation by the mirofilament cytoskeleton, and the consequences of these on early and late signaling events will be investigated in biochemical preparations and intact cells. Because of the central role of ligand binding and receptor aggregation in immune cell activation and the accumulating evidence that signaling molecules localized to membrane domains are integrally involved, the proposed investigation should lead to new concepts and targets for therapeutic intervention.
Keywords: antibody receptor, biological signal transduction, crosslink, hypersensitivity, immunoglobulin E, receptor binding, DNA, cell membrane, mast cell, membrane activity, membrane lipid, phosphorylation, protein tyrosine kinase, flow cytometry, tissue /cell culture
Project start date: 1985-08-01
Project end date: 2002-06-30
2R01AI022449-11A2 (1997): $183815
Antigen Binding, Crosslinking And Signaling
David A Holowka, Senior Scientist
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 2R01AI022449-16A1 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: The proposed research addresses key unanswered questions about the structures and events by which antigen-activated immunoreceptors initiate cellular signaling leading to diverse responses. The high affinity receptor for lgE (FcepsilonRI) on RBL-2H3 mast cells serves as a valuable model system, and new experimental thrusts will build on previous extensive studies. Specific Aim #1 extends experiments with structurally defined ligands to extract critical features of ligand architecture and cross-linking mechanism that facilitate or inhibit signaling responses. Specific Aim #2 continues efforts to understand the molecular basis by which lipid rafts participate in FcepsilonRI signal initiation, and to achieve functional reconstitution of this receptor in model membranes from purified, defined components. Specific Aim #3 proposes real-time imaging studies to elucidate the process by which FcepsilonRI becomes segregated from particular lipid raft components, and investigate how plasma membrane locations for stimulated exocytosis are determined. Specific Aim #4 proposes experiments to understand the mechanisms by which Rho family proteins and other low molecular weight GTPases participate in early FcepsilonRI signaling, as revealed by previous studies with mutant RBL-B6A4C1 mast cells. These investigations integrate diverse physical, chemical, and biological approaches to provide novel insights into the structure and molecular mechanisms of IgE-FcepsilonRI signaling, and they will advance our molecular understanding of the cell biology of this important immune cell prototype.
Keywords: antibody receptor, antigen receptor, biological signal transduction, cellular immunity, crosslink, immunoglobulin E, receptor binding, B cell receptor, T cell receptor, exocytosis, granulocyte, guanosinetriphosphatase, mast cell, phosphorylation, protein tyrosine kinase, CHO cell, flow cytometry, tissue /cell culture, western blotting
Project start date: 1985-08-01
Project end date: 2008-04-30
2R01AI022449-16A1 (2003): $260663
ANTIGEN BINDING, CROSSLINKING AND TRANSMEMBRANE SIGNALING
David A Holowka
Cornell University Ithaca, Office Of Sponsored Programs, Ithaca, Ny 14850-2820
Grant 2R01AI022449-21A2 from National Institute Of Allergy And Infectious Diseases
Abstract: Proposed work will continue our long-term efforts to understand molecular mechanisms by which crosslinking of IgE receptors on mast cells triggers complex cellular signaling processes that lead to important functional responses in immune host defense. Central to mast cell and other leukocyte signaling responses is mobilization of intracellular Ca2+, and we will investigate this spatio-temporally orchestrated process and its functional roles in mast cell exocytosis, cytokine production, and dynamic interactions with epithelial tissue. Specific Aim 1 focuses on elucidating molecular mechanisms of IgE receptor-activated Ca2+ responses, including novel antigen-stimulated Ca2+ waves that precede store-operated Ca2+ influx and impart spatial direction to this signaling response. The mechanism for activation of store-operated Ca2+ influx via recently identified Orai1/CRACM1 calcium channels will be characterized using fluorescence resonance energy transfer imaging for measurements of molecular proximity between this channel and the calcium sensor, STIM1. We will utilize this method to characterize the mechanism for regulation of Ca2+ influx by phosphatidylinositol 4,5- bisphosphate (PIP2) that we recently identified. Our evidence that different family members of phosphatidylinositol 4-phosphate 5-kinases synthesize functionally separate pools of PIP2 involved in Ca2+ mobilization leads us to propose that these PIP2 pools are spatially segregated in the plasma membrane, and we will test this hypothesis using biochemical, molecular genetic and imaging approaches. We established the participation of Ca2+ mobilization in IgE-receptor stimulated trafficking from recycling endosomes, and in Aim 2 we will test the role of this process in stimulated cytokine secretion important for adaptive immune responses. We will also test our hypothesis that protein kinase C regulates the access of secretory granules to Ca2+/PIP2/synaptotagmin complexes at the plasma membrane. In Aim 3 we will characterize cellular mechanisms for mucosal mast cell motility, chemotaxis, and dynamic interactions with intestinal epithelial cells, both in cell culture and in live tissue. Mast cell motility and interactions within physiological tissue are regulated by changes in Ca2+ levels, and our proposed work will extend our general understanding of Ca2+ mobilization as a fundamental yet complex regulator of hematopoietic cellular homeostasis and response. Mast cells play a central role in allergic immune responses, and their significant participation in innate as well adaptive immunology is increasingly appreciated. Many aspects of mast cell function depend on highly orchestrated changes in intracellular Ca2+ in response to stimulation through the IgE receptor and by other means. Our proposed research draws from a broad range of experimental approaches to investigate molecular mechanisms for Ca2+-dependent signaling in key responses, including degranulation to release chemical mediators in allergies, cytokine production to recruit other cells in inflammation, and dynamic interactions of mast cells with epithelial cells as a first line of defense to invading pathogens. Elucidation of mast cell mechanisms will provide new opportunities for intervention in therapeutic applications and a clearer understanding of hematopoietic cell biology that is seminal to regulation of immune responses in health and disease
Keywords: No Project Terms available
Relevance: Relevance to Public Health Mast cells play a central role in allergic immune responses, and their significant participation in innate as well adaptive immunology is increasingly appreciated. Many aspects of mast cell function depend on highly orchestrated changes in intracellular Ca2+ in response to stimulation through the IgE receptor and by other means. Our proposed research draws from a broad range of experimental approaches to investigate molecular mechanisms for Ca2+-dependent signaling in key responses, including degranulation to release chemical mediators in allergies, cytokine production to recruit other cells in inflammation, and dynamic interactions of mast cells with epithelial cells as a first line of defense to invading pathogens. Elucidation of mast cell mechanisms will provide new opportunities for intervention in therapeutic applications and a clearer understanding of hematopoietic cell biology that is seminal to regulation of immune responses in health and disease
Project start date: 1985-08-01
Project end date: 2014-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
2R01AI022449-21A2 (2010): $387753
2R01AI022449-08 (1993): $186441
ANTIGEN BINDING, CROSSLINKING & TRANSMEMBRANE SIGNALING
David A Holowka, Senior Scientist
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 3R01AI022449-10S2 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Project start date: 1985-08-01
Project end date: 1997-06-30
3R01AI022449-10S2 (1996): $208789