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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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Estimation of number of HUTS-21 and anti-CD49d (9F10, anti α4-integrin) mAbs binding sites on U937 cells. Cells were incubated with isotype control, HUTS-21, or 9F10 (see the “Experimental Procedures” for details). A, histograms for cell autofluorescence and isotype control (G155–178, isotype control for HUTS-21). B, histogram for HUTS-21 binding. C, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for HUTS-21 mAbs. D, histograms for cell autofluorescence and isotype control (MOPC-21, isotype control for 9F10). E, histogram for 9F10(anti-CD49d) binding. F, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for 9F10 (anti-CD49d) mAbs. Equations and calculated ABCs are shown in panels C and F. The difference in the ABC values for HUTS-21 and CD49d was not statistically significant.
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fig3: Estimation of number of HUTS-21 and anti-CD49d (9F10, anti α4-integrin) mAbs binding sites on U937 cells. Cells were incubated with isotype control, HUTS-21, or 9F10 (see the “Experimental Procedures” for details). A, histograms for cell autofluorescence and isotype control (G155–178, isotype control for HUTS-21). B, histogram for HUTS-21 binding. C, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for HUTS-21 mAbs. D, histograms for cell autofluorescence and isotype control (MOPC-21, isotype control for 9F10). E, histogram for 9F10(anti-CD49d) binding. F, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for 9F10 (anti-CD49d) mAbs. Equations and calculated ABCs are shown in panels C and F. The difference in the ABC values for HUTS-21 and CD49d was not statistically significant.

Mentions: The Number of Bound HUTS-21 Corresponds to the Total Number of CD49d Binding Sites—Next, to quantify the number HUTS-21 binding sites on U937 cells, we used Quantum Simply Cellular beads (Bangs Laboratories) to calibrate antibody binding. Cells were saturated with HUTS-21 in the presence of a saturating amount of LDV ligand (1 μm) (Fig. 3). In parallel, cells were stained with anti-CD49d (α4-integrin subunit) antibodies. Simply Cellular beads were saturated with the same antibodies. This resulted in two calibration lines that were used to estimate ABCs (Fig. 3, C and F). The data showed that the numbers of bound HUTS-21 (ABC ∼ 164,000) and anti-CD49d (ABC ∼ 179,000) were very similar, indicating that essentially every VLA-4 molecule can adopt a conformation with exposed HUTS-21 epitope after LDV ligand binding. As shown previously, the number of VLA-4 molecules detected on U937 cells is similar to the number of fluorescent LDV-FITC molecules bound to U937 cells (14). Therefore, the number of VLA-4 sites with bound HUTS-21 corresponds to the number of sites occupied by the ligand.


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)

Estimation of number of HUTS-21 and anti-CD49d (9F10, anti α4-integrin) mAbs binding sites on U937 cells. Cells were incubated with isotype control, HUTS-21, or 9F10 (see the “Experimental Procedures” for details). A, histograms for cell autofluorescence and isotype control (G155–178, isotype control for HUTS-21). B, histogram for HUTS-21 binding. C, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for HUTS-21 mAbs. D, histograms for cell autofluorescence and isotype control (MOPC-21, isotype control for 9F10). E, histogram for 9F10(anti-CD49d) binding. F, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for 9F10 (anti-CD49d) mAbs. Equations and calculated ABCs are shown in panels C and F. The difference in the ABC values for HUTS-21 and CD49d was not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Estimation of number of HUTS-21 and anti-CD49d (9F10, anti α4-integrin) mAbs binding sites on U937 cells. Cells were incubated with isotype control, HUTS-21, or 9F10 (see the “Experimental Procedures” for details). A, histograms for cell autofluorescence and isotype control (G155–178, isotype control for HUTS-21). B, histogram for HUTS-21 binding. C, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for HUTS-21 mAbs. D, histograms for cell autofluorescence and isotype control (MOPC-21, isotype control for 9F10). E, histogram for 9F10(anti-CD49d) binding. F, calibration curve generated using Simply Cellular anti-mouse IgG microspheres for 9F10 (anti-CD49d) mAbs. Equations and calculated ABCs are shown in panels C and F. The difference in the ABC values for HUTS-21 and CD49d was not statistically significant.
Mentions: The Number of Bound HUTS-21 Corresponds to the Total Number of CD49d Binding Sites—Next, to quantify the number HUTS-21 binding sites on U937 cells, we used Quantum Simply Cellular beads (Bangs Laboratories) to calibrate antibody binding. Cells were saturated with HUTS-21 in the presence of a saturating amount of LDV ligand (1 μm) (Fig. 3). In parallel, cells were stained with anti-CD49d (α4-integrin subunit) antibodies. Simply Cellular beads were saturated with the same antibodies. This resulted in two calibration lines that were used to estimate ABCs (Fig. 3, C and F). The data showed that the numbers of bound HUTS-21 (ABC ∼ 164,000) and anti-CD49d (ABC ∼ 179,000) were very similar, indicating that essentially every VLA-4 molecule can adopt a conformation with exposed HUTS-21 epitope after LDV ligand binding. As shown previously, the number of VLA-4 molecules detected on U937 cells is similar to the number of fluorescent LDV-FITC molecules bound to U937 cells (14). Therefore, the number of VLA-4 sites with bound HUTS-21 corresponds to the number of sites occupied by the ligand.

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