. Integrins are cell surface receptors that mediate numerous interactions between cells and their environment. Binding of integrin ?5?1 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 ?5?1 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 ?5?1 state. Using the same Fab tools as used for these equilibrium measurements to stabilize the extended, closed, or open ?5?1 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 ?5?1 is so stable in the BC state, we test the hypothesis that the ? and ?-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 ?5?1, i.e. the sequence of ligand binding and conformational change events that occur between the resting state, when 99.8% of unliganded integrin ?5?1 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.

Public Health Relevance

. Integrin ?5?1 is a cell surface protein that directs how cells interact with both other cells and molecules within the extracellular matrix. This grant will explore how different factors change the conformational shape of integrin ?5?1, and how those changes in shape affect its ability to interact with an important protein binding partner called fibronectin in blood cells. Insights from this research will shed light on how integrin ?5?1 and other related integrins function and will aid the development of drugs to treat diseases that are both dependent on, and exacerbated by integrin function including atherosclerosis, fibrosis, sickle cell disease, and cancer.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Intercellular Interactions Study Section (ICI)
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Kindzelski, Andrei L
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Boston Children's Hospital
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Li, Jing; Springer, Timothy A (2017) Integrin extension enables ultrasensitive regulation by cytoskeletal force. Proc Natl Acad Sci U S A 114:4685-4690
Li, Jing; Su, Yang; Xia, Wei et al. (2017) Conformational equilibria and intrinsic affinities define integrin activation. EMBO J 36:629-645
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