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Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation.

Chigaev A, Waller A, Amit O, Halip L, Bologa CG, Sklar LA - J. Biol. Chem. (2009)

Bottom Line: We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope.Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding.Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. achigaev@salud.unm.edu

ABSTRACT
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC(50) for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

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Kinetics of real-time binding of HUTS-21 antibodies to U937 cells, transfected with a non-desensitizing mutant of formyl peptide receptor. A, cells were treated with 100 nm of fMLFF (activated) or DMSO (vehicle) 5 min before the start of the experiment. The addition of HUTS-21 antibodies (first arrow) resulted in a rapid nonspecific binding of the antibody. Next, increasing amounts of LDV ligand were added. This induced binding of mAbs and resulted in different rates of antibody binding (compare slopes after LDV additions). B, absolute rates HUTS-21 binding (slopes of lines between sequential LDV additions calculated from panel A) plotted versus concentration of LDV in solution. The fit to the data was done using the sigmoidal dose-response equation with variable slope using GraphPad Prism software. Difference in EC50 values for resting and activated cells indicated the affinity change for LDV binding. A representative experiment of three independent experiments is shown. C, the slopes of the lines between sequential LDV additions, calculated from panel A, are plotted versus the fraction of VLA-4 occupied by LDV. The fraction of VLA-4 occupied by LDV was calculated using the one-site binding equation (Y = 100 × LDV/Kd + LDV, where Y is % of sites occupied, LDV is LDV concentration, and Kd is a previously published dissociation constant for resting and fMLFF activated states). D, the change in the rates of HUTS-21 binding can be seen in real-time. Cells pretreated with HUTS-21 antibodies (first arrow) were treated with a very high saturating concentration of LDV (10 μm, second arrow). Next, cells were activated by fMLFF. Control samples (DMSO) are also shown. Despite the fact that VLA-4 is completely saturated by LDV (10 μm is ∼1000-fold higher than Kd), the change in the slope of the line indicating HUTS-21 binding can be detected. A representative experiment of three independent experiments is shown.
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fig4: Kinetics of real-time binding of HUTS-21 antibodies to U937 cells, transfected with a non-desensitizing mutant of formyl peptide receptor. A, cells were treated with 100 nm of fMLFF (activated) or DMSO (vehicle) 5 min before the start of the experiment. The addition of HUTS-21 antibodies (first arrow) resulted in a rapid nonspecific binding of the antibody. Next, increasing amounts of LDV ligand were added. This induced binding of mAbs and resulted in different rates of antibody binding (compare slopes after LDV additions). B, absolute rates HUTS-21 binding (slopes of lines between sequential LDV additions calculated from panel A) plotted versus concentration of LDV in solution. The fit to the data was done using the sigmoidal dose-response equation with variable slope using GraphPad Prism software. Difference in EC50 values for resting and activated cells indicated the affinity change for LDV binding. A representative experiment of three independent experiments is shown. C, the slopes of the lines between sequential LDV additions, calculated from panel A, are plotted versus the fraction of VLA-4 occupied by LDV. The fraction of VLA-4 occupied by LDV was calculated using the one-site binding equation (Y = 100 × LDV/Kd + LDV, where Y is % of sites occupied, LDV is LDV concentration, and Kd is a previously published dissociation constant for resting and fMLFF activated states). D, the change in the rates of HUTS-21 binding can be seen in real-time. Cells pretreated with HUTS-21 antibodies (first arrow) were treated with a very high saturating concentration of LDV (10 μm, second arrow). Next, cells were activated by fMLFF. Control samples (DMSO) are also shown. Despite the fact that VLA-4 is completely saturated by LDV (10 μm is ∼1000-fold higher than Kd), the change in the slope of the line indicating HUTS-21 binding can be detected. A representative experiment of three independent experiments is shown.

Mentions: The Rate of HUTS-21 Binding Can Be Used to Determine Integrin Affinity State—According to our data, binding of HUTS-21 can be described in the following simple model, (Eq. 1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\;\;L+R\begin{matrix}k_{+1}\\ {\longleftrightarrow}\\ k_{-1}\end{matrix}LR\;\;\end{equation*}\end{document} (Eq. 2)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\;\;LR+HUTS\begin{matrix}k_{+2}\\ {\rightarrow}\\ \end{matrix}(LR{\cdot}HUTS)\;\;\end{equation*}\end{document} where L is the concentration of LDV ligand, R is the concentration of VLA-4 receptor, and LR is the concentration of ligand-receptor complex. HUTS is the concentration of HUTS antibodies, and LR·HUTS is the concentration of HUTS bound to ligand-occupied VLA-4. Because flow cytometers have the ability to discriminate between free and bound fluorescent ligand in a homogeneous assay (18), MCF is proportional to LR·HUTS. In the absence of LDV ligand, no ligand receptor complex is formed, and therefore, no binding of HUTS-21 is observed (Fig. 1, B and C, DMSO line). The addition of different concentrations of LDV results in the formation of the ligand-receptor complex, and this is reflected in the different rates of HUTS-21 binding (Figs. 2A and 4A). Because binding of a large antibody molecule is limited by diffusion, antibody binding is slow compared with the binding of a small LDV molecule. Also on this time scale binding of antibodies is virtually irreversible (Equation 2). The ligand equilibration time (and approach to equilibrium) is determined by its dissociation rate constant (k-1). For the LDV ligand, k-1 varies from 0.1–0.06 s-1 for the resting state to 0.02–0.01 s-1 for fMLFF-activated state (14, 15). Therefore, for these affinity states (resting and fMLFF activated) equilibrium is reached within a few minutes after ligand addition (see Refs. 14 and 15 for real-time LDV-FITC binding kinetics). Binding of HUTS-21 on this time scale is very far from equilibrium (see Fig. 1C), and therefore, binding of HUTS is represented by a series of straight lines (Figs. 2A and 4A). Thus, under these conditions, the binding rate of HUTS-21 is determined by the amount of LR at each time (Equations 1 and 2). Therefore, the observed rate of HUTS-21 binding (the slope of the line, Figs. 2A and 4A) is proportional to the concentration of ligand receptor complex at each time.


Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation.

Chigaev A, Waller A, Amit O, Halip L, Bologa CG, Sklar LA - J. Biol. Chem. (2009)

Kinetics of real-time binding of HUTS-21 antibodies to U937 cells, transfected with a non-desensitizing mutant of formyl peptide receptor. A, cells were treated with 100 nm of fMLFF (activated) or DMSO (vehicle) 5 min before the start of the experiment. The addition of HUTS-21 antibodies (first arrow) resulted in a rapid nonspecific binding of the antibody. Next, increasing amounts of LDV ligand were added. This induced binding of mAbs and resulted in different rates of antibody binding (compare slopes after LDV additions). B, absolute rates HUTS-21 binding (slopes of lines between sequential LDV additions calculated from panel A) plotted versus concentration of LDV in solution. The fit to the data was done using the sigmoidal dose-response equation with variable slope using GraphPad Prism software. Difference in EC50 values for resting and activated cells indicated the affinity change for LDV binding. A representative experiment of three independent experiments is shown. C, the slopes of the lines between sequential LDV additions, calculated from panel A, are plotted versus the fraction of VLA-4 occupied by LDV. The fraction of VLA-4 occupied by LDV was calculated using the one-site binding equation (Y = 100 × LDV/Kd + LDV, where Y is % of sites occupied, LDV is LDV concentration, and Kd is a previously published dissociation constant for resting and fMLFF activated states). D, the change in the rates of HUTS-21 binding can be seen in real-time. Cells pretreated with HUTS-21 antibodies (first arrow) were treated with a very high saturating concentration of LDV (10 μm, second arrow). Next, cells were activated by fMLFF. Control samples (DMSO) are also shown. Despite the fact that VLA-4 is completely saturated by LDV (10 μm is ∼1000-fold higher than Kd), the change in the slope of the line indicating HUTS-21 binding can be detected. A representative experiment of three independent experiments is shown.
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fig4: Kinetics of real-time binding of HUTS-21 antibodies to U937 cells, transfected with a non-desensitizing mutant of formyl peptide receptor. A, cells were treated with 100 nm of fMLFF (activated) or DMSO (vehicle) 5 min before the start of the experiment. The addition of HUTS-21 antibodies (first arrow) resulted in a rapid nonspecific binding of the antibody. Next, increasing amounts of LDV ligand were added. This induced binding of mAbs and resulted in different rates of antibody binding (compare slopes after LDV additions). B, absolute rates HUTS-21 binding (slopes of lines between sequential LDV additions calculated from panel A) plotted versus concentration of LDV in solution. The fit to the data was done using the sigmoidal dose-response equation with variable slope using GraphPad Prism software. Difference in EC50 values for resting and activated cells indicated the affinity change for LDV binding. A representative experiment of three independent experiments is shown. C, the slopes of the lines between sequential LDV additions, calculated from panel A, are plotted versus the fraction of VLA-4 occupied by LDV. The fraction of VLA-4 occupied by LDV was calculated using the one-site binding equation (Y = 100 × LDV/Kd + LDV, where Y is % of sites occupied, LDV is LDV concentration, and Kd is a previously published dissociation constant for resting and fMLFF activated states). D, the change in the rates of HUTS-21 binding can be seen in real-time. Cells pretreated with HUTS-21 antibodies (first arrow) were treated with a very high saturating concentration of LDV (10 μm, second arrow). Next, cells were activated by fMLFF. Control samples (DMSO) are also shown. Despite the fact that VLA-4 is completely saturated by LDV (10 μm is ∼1000-fold higher than Kd), the change in the slope of the line indicating HUTS-21 binding can be detected. A representative experiment of three independent experiments is shown.
Mentions: The Rate of HUTS-21 Binding Can Be Used to Determine Integrin Affinity State—According to our data, binding of HUTS-21 can be described in the following simple model, (Eq. 1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\;\;L+R\begin{matrix}k_{+1}\\ {\longleftrightarrow}\\ k_{-1}\end{matrix}LR\;\;\end{equation*}\end{document} (Eq. 2)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\;\;LR+HUTS\begin{matrix}k_{+2}\\ {\rightarrow}\\ \end{matrix}(LR{\cdot}HUTS)\;\;\end{equation*}\end{document} where L is the concentration of LDV ligand, R is the concentration of VLA-4 receptor, and LR is the concentration of ligand-receptor complex. HUTS is the concentration of HUTS antibodies, and LR·HUTS is the concentration of HUTS bound to ligand-occupied VLA-4. Because flow cytometers have the ability to discriminate between free and bound fluorescent ligand in a homogeneous assay (18), MCF is proportional to LR·HUTS. In the absence of LDV ligand, no ligand receptor complex is formed, and therefore, no binding of HUTS-21 is observed (Fig. 1, B and C, DMSO line). The addition of different concentrations of LDV results in the formation of the ligand-receptor complex, and this is reflected in the different rates of HUTS-21 binding (Figs. 2A and 4A). Because binding of a large antibody molecule is limited by diffusion, antibody binding is slow compared with the binding of a small LDV molecule. Also on this time scale binding of antibodies is virtually irreversible (Equation 2). The ligand equilibration time (and approach to equilibrium) is determined by its dissociation rate constant (k-1). For the LDV ligand, k-1 varies from 0.1–0.06 s-1 for the resting state to 0.02–0.01 s-1 for fMLFF-activated state (14, 15). Therefore, for these affinity states (resting and fMLFF activated) equilibrium is reached within a few minutes after ligand addition (see Refs. 14 and 15 for real-time LDV-FITC binding kinetics). Binding of HUTS-21 on this time scale is very far from equilibrium (see Fig. 1C), and therefore, binding of HUTS is represented by a series of straight lines (Figs. 2A and 4A). Thus, under these conditions, the binding rate of HUTS-21 is determined by the amount of LR at each time (Equations 1 and 2). Therefore, the observed rate of HUTS-21 binding (the slope of the line, Figs. 2A and 4A) is proportional to the concentration of ligand receptor complex at each time.

Bottom Line: We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope.Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding.Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. achigaev@salud.unm.edu

ABSTRACT
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC(50) for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

Show MeSH
Related in: MedlinePlus