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Principal Investigator: TIMOTHY A SPRINGER
Organization: BOSTON CHILDREN'S HOSPITAL
Fiscal Year: 2024
Award: $719,617
Funding agency: National Heart Lung and Blood Institute
Abstract.
Integrins are cell surface receptors that mediate numerous interactions between cells and their environment.
Binding of integrin α5β1 to its ligand fibronectin in the extracellular matrix plays fundamental roles in cellular
adhesion and differentiation. In blood cells these interactions mediate biological processes including
erythropoiesis and monocyte adhesion and contribute to pathologies such as sickle cell disease, dysregulation
of hematopoiesis, atherosclerosis, and fibrosis. Integrin α5β1 undergoes two distinct conformational changes:
extension at the ‘knees’ and opening of the ligand-binding headpiece. These changes give rise to an ensemble
of three interconverting integrin conformational states on cell surfaces: low-affinity bent-closed (BC) and
extended-closed (EC) conformations and a high-affinity extended-open (EO) conformation. This proposal
leverages ground-breaking work under the previous award in which we measured free energy and intrinsic
affinity of each integrin α5β1 state. Using the same Fab tools as used for these equilibrium measurements to
stabilize the extended, closed, or open α5β1 conformations, we now propose three aims. In Aim 1, we explore
how Mn2+, high Mg2+, and low Ca2+ concentrations activate integrins. Our preliminary results show that Mn2+
and high Mg2+ both increase the population of the EO state and increase its intrinsic affinity for ligand and that
these effects are dependent on the ADMIDAS metal-ion binding site. To examine why cell surface α5β1 is so
stable in the BC state, we test the hypothesis that the α and β-subunit TM domains separate from one another
in both the EC and EO states. Aims 2 and 3 measure kinetics to map the activation trajectory of integrin α5β1,
i.e. the sequence of ligand binding and conformational change events that occur between the resting state,
when 99.8% of unliganded integrin α5β1 is in the BC state, and the final, functional liganded EO state (EO•L)
state that is bound to fibronectin and is stabilized by tensile force that is applied to the integrin by actin
retrograde flow and resisted by fibronectin in the matrix. In Aim 2, we measure the intrinsic ligand-binding
kinetics of each state (kon and koff). Our preliminary data indicates, surprisingly, that the low-affinity BC and EC
states bind more rapidly to ligand than the EO state, which is compensated by the >10,000-fold slower off-rate
of the EO state. In Aim 3, we measure the kinetics of integrin conformational change using single-molecule
FRET probes that measure either the extension or opening steps in the presence or absence of conformation-
specific Fabs and ligand. Kinetics of all transitions between the BC, EC, and EO states for unliganded and
ligand-bound single integrin molecules will be determined for both purified, soluble ectodomain and intact
integrins on blood cells using TIRF microscopy with high temporal resolution. We expect to show an integrin
activation trajectory in which ligand binds to the BC+EC states, followed by ligand-facilitated conformational
conversion to the EO•L state, followed by cytoskeletal adaptor (A) binding and stabilization of the EO•L•A state
by force applied by the cytoskeleton and resisted by extracellular ligand.
Terms: <Acceleration><Actins><Adhesions><Affect><Affinity><Amino Acids><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Award><Binding><Binding Proteins><Binding Sites><Biological Function><Biological Process><Blood Cells><Blood monocyte><Body Tissues><Cancers><Cell Adhesion><Cell Body><Cell Communication><Cell Differentiation><Cell Differentiation process><Cell Interaction><Cell Locomotion><Cell Migration><Cell Movement><Cell Surface Proteins><Cell Surface Receptors><Cell surface><Cell-Extracellular Matrix><Cell-to-Cell Interaction><Cells><Cellular Adhesion><Cellular Matrix><Cellular Migration><Cellular Motility><Cold-Insoluble Globulins><Combining Site><Compensation><Coupling><Cy5><Cytoskeletal System><Cytoskeleton><Data><Disease><Disorder><Disputes><Dissociation><Disulfides><Divalent Cations><ECM><Environment><Equilibrium><Erythropoiesis><Event><Extracellular Matrix><FN1><FRET><Fibronectin 1><Fibronectins><Fibrosis><Fluorescence Resonance Energy Transfer><Free Energy><Förster Resonance Energy Transfer><Genetic Alteration><Genetic Change><Genetic defect><Grant><Hb SS disease><HbSS disease><Hematopoiesis><Hematopoietic Cellular Control Mechanisms><Hemoglobin S Disease><Hemoglobin sickle cell disease><Hemoglobin sickle cell disorder><Integrin Binding><Integrin alpha-5 beta-1><Integrin alpha5beta1><Integrin beta Chains><Integrin beta Subunits><Integrin α5β1><Integrin β Chains><Integrin β Subunits><Integrins><Integrins Extracellular Matrix><Ions><Kinetics><Knee><LETS Proteins><Large External Transformation-Sensitive Protein><Ligand Binding><Ligand Binding Protein><Ligand Binding Protein Gene><Ligands><Link><Malignant Neoplasms><Malignant Tumor><Maps><Marrow monocyte><Measurement><Measures><Mediating><Membrane><Metal Ion Binding><Metals><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Mutate><Mutation><Opsonic Glycoprotein><Opsonic alpha(2)SB Glycoprotein><Pathology><Peripheral Blood Cell><Platelet Glycoprotein Ic/IIa><Play><Population><Position><Positioning Attribute><Protein Binding><Reaction><Reactive Site><Reporting><Research><Rest><Role><Shapes><Sickle Cell Anemia><Site><Structure><Supporting Cell><TIRF Microscopy><TM Domain><Testing><Tissues><Titrations><Total Internal Reflection Fluorescent><Total Internal Reflection Fluorescent Microscopy><Transmembrane Domain><Transmembrane Region><VLA-5><VLA-5 Receptors><Work><alpha 2-Surface Binding Glycoprotein><aminoacid><atheromatosis><atherosclerotic disease><atherosclerotic vascular disease><balance><balance function><beta Integrins><blood cell formation><bound protein><cell motility><cellular differentiation><conformation><conformational><conformational conversion><conformational state><conformational transition><conformationally><conformations><cyanine dye 5><drug development><erythroid development><experiment><experimental research><experimental study><experiments><extracellular><fluorophore><genome mutation><insight><integrin bound><intracellular skeleton><malignancy><membrane structure><monocyte><neoplasm/cancer><rate of change><sickle cell disease><sickle cell disorder><sickle disease><sicklemia><single molecule><social role><temporal measurement><temporal resolution><time measurement><tool><β-Integrins>