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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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Detection of high affinity state of VLA-4 using HUTS-21 antibodies. A, simulation of LDV binding for two affinity states of VLA-4 performed using a sigmoidal dose-response binding equation (Y = 1/(1 + 10(log EC50 - X)), where Y is receptor occupancy, and X is the log of LDV concentration). EC50 values for resting and activated states are indicated. The maximal difference receptor occupancy for low and high affinity states was determined by subtracting the resting curve from the activated curve on a point by point basis. The maximal difference was observed at log -8.5 = 3.2 nm of LDV in solution. The arrow indicates additional binding of LDV ligand at 3.2 nm from low affinity receptor state (point 1) to a high affinity receptor state (point 2). B, experimental data showing additional binding of HUTS-21 antibodies induced by fMLFF activation in the presence of 3.2 nm LDV. Histograms for cell autofluorescence, isotype control, low affinity state (LDV + DMSO), and high affinity state (LDV + fMLFF) are shown. Each histogram represents a mean of three independent determinations (n = 3). A representative experiment of three independent experiments is shown.
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fig5: Detection of high affinity state of VLA-4 using HUTS-21 antibodies. A, simulation of LDV binding for two affinity states of VLA-4 performed using a sigmoidal dose-response binding equation (Y = 1/(1 + 10(log EC50 - X)), where Y is receptor occupancy, and X is the log of LDV concentration). EC50 values for resting and activated states are indicated. The maximal difference receptor occupancy for low and high affinity states was determined by subtracting the resting curve from the activated curve on a point by point basis. The maximal difference was observed at log -8.5 = 3.2 nm of LDV in solution. The arrow indicates additional binding of LDV ligand at 3.2 nm from low affinity receptor state (point 1) to a high affinity receptor state (point 2). B, experimental data showing additional binding of HUTS-21 antibodies induced by fMLFF activation in the presence of 3.2 nm LDV. Histograms for cell autofluorescence, isotype control, low affinity state (LDV + DMSO), and high affinity state (LDV + fMLFF) are shown. Each histogram represents a mean of three independent determinations (n = 3). A representative experiment of three independent experiments is shown.

Mentions: HUTS-21 Can Be Used to Detect the Activated (High Affinity) State of VLA-4—Previously we established a simple method for the detection of VLA-4 affinity change in real time on live cells (14). A transition from low to high affinity state leads to additional binding of the LDV-FITC probe if cells were preincubated with the ligand at concentrations below the Kd for the low affinity state and above the Kd for the high affinity state. In the case of a fluorescent ligand this additional binding can be detected using a conventional flow cytometer in a homogeneous assay (4, 14, 15). Our present data show that binding of HUTS-21 antibodies can be used as a reporter of a ligand-occupied receptor (see Fig. 2B). Therefore, detection of the high affinity state of VLA-4 can be performed with an unlabeled ligand and HUTS-21. To determine the concentration where the biggest difference in the ligand binding after activation will be observed, we generated two theoretical binding curves (Fig. 5). Next, we have determined that at 3.2 nm (log -8.5) VLA-4 receptor occupancy would increase from ∼0.24 to ∼0.76 after the affinity change (arrow, Fig. 5A). The experiment, where HUTS-21 binding occurred in the presence of 3.2 nm LDV, showed a significant difference between resting and fMLFF-activated cells (Fig. 5B, compare LDV + DMSO (control) with LDV + fMLFF). Thus, in the presence of properly chosen concentration of the ligand (∼3.2 nm for LDV), binding of HUTS-21 is also sensitive to cellular activation status.


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)

Detection of high affinity state of VLA-4 using HUTS-21 antibodies. A, simulation of LDV binding for two affinity states of VLA-4 performed using a sigmoidal dose-response binding equation (Y = 1/(1 + 10(log EC50 - X)), where Y is receptor occupancy, and X is the log of LDV concentration). EC50 values for resting and activated states are indicated. The maximal difference receptor occupancy for low and high affinity states was determined by subtracting the resting curve from the activated curve on a point by point basis. The maximal difference was observed at log -8.5 = 3.2 nm of LDV in solution. The arrow indicates additional binding of LDV ligand at 3.2 nm from low affinity receptor state (point 1) to a high affinity receptor state (point 2). B, experimental data showing additional binding of HUTS-21 antibodies induced by fMLFF activation in the presence of 3.2 nm LDV. Histograms for cell autofluorescence, isotype control, low affinity state (LDV + DMSO), and high affinity state (LDV + fMLFF) are shown. Each histogram represents a mean of three independent determinations (n = 3). A representative experiment of three independent experiments is shown.
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Related In: Results  -  Collection

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fig5: Detection of high affinity state of VLA-4 using HUTS-21 antibodies. A, simulation of LDV binding for two affinity states of VLA-4 performed using a sigmoidal dose-response binding equation (Y = 1/(1 + 10(log EC50 - X)), where Y is receptor occupancy, and X is the log of LDV concentration). EC50 values for resting and activated states are indicated. The maximal difference receptor occupancy for low and high affinity states was determined by subtracting the resting curve from the activated curve on a point by point basis. The maximal difference was observed at log -8.5 = 3.2 nm of LDV in solution. The arrow indicates additional binding of LDV ligand at 3.2 nm from low affinity receptor state (point 1) to a high affinity receptor state (point 2). B, experimental data showing additional binding of HUTS-21 antibodies induced by fMLFF activation in the presence of 3.2 nm LDV. Histograms for cell autofluorescence, isotype control, low affinity state (LDV + DMSO), and high affinity state (LDV + fMLFF) are shown. Each histogram represents a mean of three independent determinations (n = 3). A representative experiment of three independent experiments is shown.
Mentions: HUTS-21 Can Be Used to Detect the Activated (High Affinity) State of VLA-4—Previously we established a simple method for the detection of VLA-4 affinity change in real time on live cells (14). A transition from low to high affinity state leads to additional binding of the LDV-FITC probe if cells were preincubated with the ligand at concentrations below the Kd for the low affinity state and above the Kd for the high affinity state. In the case of a fluorescent ligand this additional binding can be detected using a conventional flow cytometer in a homogeneous assay (4, 14, 15). Our present data show that binding of HUTS-21 antibodies can be used as a reporter of a ligand-occupied receptor (see Fig. 2B). Therefore, detection of the high affinity state of VLA-4 can be performed with an unlabeled ligand and HUTS-21. To determine the concentration where the biggest difference in the ligand binding after activation will be observed, we generated two theoretical binding curves (Fig. 5). Next, we have determined that at 3.2 nm (log -8.5) VLA-4 receptor occupancy would increase from ∼0.24 to ∼0.76 after the affinity change (arrow, Fig. 5A). The experiment, where HUTS-21 binding occurred in the presence of 3.2 nm LDV, showed a significant difference between resting and fMLFF-activated cells (Fig. 5B, compare LDV + DMSO (control) with LDV + fMLFF). Thus, in the presence of properly chosen concentration of the ligand (∼3.2 nm for LDV), binding of HUTS-21 is also sensitive to cellular activation status.

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