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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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LDV concentration-dependent binding of HUTS-21 to resting cells (without FPR activation). A, kinetics of real-time binding of HUTS-21 antibodies to U937 cells. The addition of HUTS-21 antibodies (first arrow) resulted in rapid nonspecific binding of antibodies. The addition of increasing amounts of LDV ligand resulted in the different rates of antibody binding (compare slopes after LDV additions). B, binding of HUTS-21 plotted versus LDV concentration. Cells were incubated with the indicated concentrations of LDV in the presence of an excess of HUTS-21 mAbs and washed, and MCF was measured. Each point represents the mean ± S.E. of three independent determinations (n = 3). The data were fitted using the sigmoidal dose-response equation with variable slope using GraphPad Prism software (the HillSlope was found to be ∼1). A representative experiment of three independent experiments is shown.
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fig2: LDV concentration-dependent binding of HUTS-21 to resting cells (without FPR activation). A, kinetics of real-time binding of HUTS-21 antibodies to U937 cells. The addition of HUTS-21 antibodies (first arrow) resulted in rapid nonspecific binding of antibodies. The addition of increasing amounts of LDV ligand resulted in the different rates of antibody binding (compare slopes after LDV additions). B, binding of HUTS-21 plotted versus LDV concentration. Cells were incubated with the indicated concentrations of LDV in the presence of an excess of HUTS-21 mAbs and washed, and MCF was measured. Each point represents the mean ± S.E. of three independent determinations (n = 3). The data were fitted using the sigmoidal dose-response equation with variable slope using GraphPad Prism software (the HillSlope was found to be ∼1). A representative experiment of three independent experiments is shown.

Mentions: Occupancy of the Ligand Binding Site in the Absence of Integrin Activation Is Sufficient to Induce HUTS Epitope Exposure—To find out how occupancy of the ligand binding site affects HUTS-21 binding, we have studied HUTS-21 binding at different concentrations of LDV ligand (Fig. 2). The addition of different concentrations of LDV resulted in a different rates of HUTS-21 binding (Fig. 2A). Long term incubation of U937 cells with a large excess of HUTS-21 in the presence of different concentrations of LDV (Fig. 2B) resulted in a sigmoidal binding curve. The EC50 for HUTS-21 binding in this case (EC50 = 11.4 nm) is identical to a previously published Kd for binding of LDV-FITC to resting U937 cells (Kd ∼ 12 nm) (14). Thus, in the absence of cell activation, LDV to the VLA-4 integrin induces HUTS-21 epitope exposure detected by HUTS-21 binding. Because the EC50 for HUTS-21 binding induced by the binding of LDV is equivalent to the Kd for ligand binding in the absence of HUTS-21, these data also confirm that binding of HUTS-21 to the β1-integrin subunit had no effect upon affinity of LDV ligand binding.


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)

LDV concentration-dependent binding of HUTS-21 to resting cells (without FPR activation). A, kinetics of real-time binding of HUTS-21 antibodies to U937 cells. The addition of HUTS-21 antibodies (first arrow) resulted in rapid nonspecific binding of antibodies. The addition of increasing amounts of LDV ligand resulted in the different rates of antibody binding (compare slopes after LDV additions). B, binding of HUTS-21 plotted versus LDV concentration. Cells were incubated with the indicated concentrations of LDV in the presence of an excess of HUTS-21 mAbs and washed, and MCF was measured. Each point represents the mean ± S.E. of three independent determinations (n = 3). The data were fitted using the sigmoidal dose-response equation with variable slope using GraphPad Prism software (the HillSlope was found to be ∼1). A representative experiment of three independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682882&req=5

fig2: LDV concentration-dependent binding of HUTS-21 to resting cells (without FPR activation). A, kinetics of real-time binding of HUTS-21 antibodies to U937 cells. The addition of HUTS-21 antibodies (first arrow) resulted in rapid nonspecific binding of antibodies. The addition of increasing amounts of LDV ligand resulted in the different rates of antibody binding (compare slopes after LDV additions). B, binding of HUTS-21 plotted versus LDV concentration. Cells were incubated with the indicated concentrations of LDV in the presence of an excess of HUTS-21 mAbs and washed, and MCF was measured. Each point represents the mean ± S.E. of three independent determinations (n = 3). The data were fitted using the sigmoidal dose-response equation with variable slope using GraphPad Prism software (the HillSlope was found to be ∼1). A representative experiment of three independent experiments is shown.
Mentions: Occupancy of the Ligand Binding Site in the Absence of Integrin Activation Is Sufficient to Induce HUTS Epitope Exposure—To find out how occupancy of the ligand binding site affects HUTS-21 binding, we have studied HUTS-21 binding at different concentrations of LDV ligand (Fig. 2). The addition of different concentrations of LDV resulted in a different rates of HUTS-21 binding (Fig. 2A). Long term incubation of U937 cells with a large excess of HUTS-21 in the presence of different concentrations of LDV (Fig. 2B) resulted in a sigmoidal binding curve. The EC50 for HUTS-21 binding in this case (EC50 = 11.4 nm) is identical to a previously published Kd for binding of LDV-FITC to resting U937 cells (Kd ∼ 12 nm) (14). Thus, in the absence of cell activation, LDV to the VLA-4 integrin induces HUTS-21 epitope exposure detected by HUTS-21 binding. Because the EC50 for HUTS-21 binding induced by the binding of LDV is equivalent to the Kd for ligand binding in the absence of HUTS-21, these data also confirm that binding of HUTS-21 to the β1-integrin subunit had no effect upon affinity of LDV ligand binding.

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