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The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2.

Kim M, Carman CV, Yang W, Salas A, Springer TA - J. Cell Biol. (2004)

Bottom Line: Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration.Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand.Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

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Affinity regulation, but not valency regulation, depends on conformational communication within the extracellular domain of LFA-1 as shown with both primary T lymphocytes and K562 transfectants. V-bottom adhesion assays were with primary human T lymphocytes (A), or with K562 cells stably expressing either wild-type αLβ2 (B) or αL-E310A/β2 (C). T lymphocytes were preincubated with 1 mM Mn2+, 1 μM PMA, 1 nM cytochalasin D, or 100 nM latrunculin A, and K562 cells were preincubated with 1 mM Mn2+, 1 μM PMA, 1 μM cytochalasin D, or 1 μM latrunculin A. Assays were in the presence of 20 μM BIRT377, 1 μM XVA143, or an equivalent concentration of DMSO as control. Data are mean ± SEM of three experiments, each in duplicate or triplicate.
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fig8: Affinity regulation, but not valency regulation, depends on conformational communication within the extracellular domain of LFA-1 as shown with both primary T lymphocytes and K562 transfectants. V-bottom adhesion assays were with primary human T lymphocytes (A), or with K562 cells stably expressing either wild-type αLβ2 (B) or αL-E310A/β2 (C). T lymphocytes were preincubated with 1 mM Mn2+, 1 μM PMA, 1 nM cytochalasin D, or 100 nM latrunculin A, and K562 cells were preincubated with 1 mM Mn2+, 1 μM PMA, 1 μM cytochalasin D, or 1 μM latrunculin A. Assays were in the presence of 20 μM BIRT377, 1 μM XVA143, or an equivalent concentration of DMSO as control. Data are mean ± SEM of three experiments, each in duplicate or triplicate.

Mentions: To extend our study on the relationship between integrin conformational change and valency regulation to nontransfected, primary T lymphocytes, we assessed the effects of two distinct classes of small molecule allosteric LFA-1 antagonists (Shimaoka and Springer, 2003). The α I allosteric antagonists, exemplified here with BIRT377, bind to the αL I domain and stabilize it in the low affinity, closed conformation. By contrast, the α/β I-like allosteric antagonists, exemplified here with XVA143, bind to the MIDAS of the β2 I-like domain and also in part to the αL subunit, and block the communication of conformational signals from the β2 I-like domain to the αL I domain (Shimaoka and Springer, 2003). Because of these different modes of action, we hypothesized that α I allosteric antagonists would inhibit both affinity and valency regulation of LFA-1, whereas α/β I-like allosteric antagonists would only inhibit affinity regulation. Indeed, the α I allosteric antagonist BIRT377 totally abolished adhesion to ICAM-1 induced by all agents tested (i.e., Mn2+, PMA, cytochalasin D, and latrunculin A), both with cultured primary human T lymphocytes (Fig. 8 A) and K562 transfectants expressing wild-type αLβ2 (Fig. 8 B). By contrast, XVA143 effectively blocked adhesion stimulated by Mn2+ and PMA, but did not inhibit adhesion stimulated by cytochalasin D or latrunculin A (Fig. 8, A and B). These results demonstrate that PMA- and Mn2+-stimulated adhesion (but not cytochalasin D– and latrunculin A–stimulated adhesion) requires conformational communication between the αL I- and β2 I-like domains, and that in all cases, the αL I domain must transit out of the closed, low affinity conformation to support adhesion.


The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2.

Kim M, Carman CV, Yang W, Salas A, Springer TA - J. Cell Biol. (2004)

Affinity regulation, but not valency regulation, depends on conformational communication within the extracellular domain of LFA-1 as shown with both primary T lymphocytes and K562 transfectants. V-bottom adhesion assays were with primary human T lymphocytes (A), or with K562 cells stably expressing either wild-type αLβ2 (B) or αL-E310A/β2 (C). T lymphocytes were preincubated with 1 mM Mn2+, 1 μM PMA, 1 nM cytochalasin D, or 100 nM latrunculin A, and K562 cells were preincubated with 1 mM Mn2+, 1 μM PMA, 1 μM cytochalasin D, or 1 μM latrunculin A. Assays were in the presence of 20 μM BIRT377, 1 μM XVA143, or an equivalent concentration of DMSO as control. Data are mean ± SEM of three experiments, each in duplicate or triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172602&req=5

fig8: Affinity regulation, but not valency regulation, depends on conformational communication within the extracellular domain of LFA-1 as shown with both primary T lymphocytes and K562 transfectants. V-bottom adhesion assays were with primary human T lymphocytes (A), or with K562 cells stably expressing either wild-type αLβ2 (B) or αL-E310A/β2 (C). T lymphocytes were preincubated with 1 mM Mn2+, 1 μM PMA, 1 nM cytochalasin D, or 100 nM latrunculin A, and K562 cells were preincubated with 1 mM Mn2+, 1 μM PMA, 1 μM cytochalasin D, or 1 μM latrunculin A. Assays were in the presence of 20 μM BIRT377, 1 μM XVA143, or an equivalent concentration of DMSO as control. Data are mean ± SEM of three experiments, each in duplicate or triplicate.
Mentions: To extend our study on the relationship between integrin conformational change and valency regulation to nontransfected, primary T lymphocytes, we assessed the effects of two distinct classes of small molecule allosteric LFA-1 antagonists (Shimaoka and Springer, 2003). The α I allosteric antagonists, exemplified here with BIRT377, bind to the αL I domain and stabilize it in the low affinity, closed conformation. By contrast, the α/β I-like allosteric antagonists, exemplified here with XVA143, bind to the MIDAS of the β2 I-like domain and also in part to the αL subunit, and block the communication of conformational signals from the β2 I-like domain to the αL I domain (Shimaoka and Springer, 2003). Because of these different modes of action, we hypothesized that α I allosteric antagonists would inhibit both affinity and valency regulation of LFA-1, whereas α/β I-like allosteric antagonists would only inhibit affinity regulation. Indeed, the α I allosteric antagonist BIRT377 totally abolished adhesion to ICAM-1 induced by all agents tested (i.e., Mn2+, PMA, cytochalasin D, and latrunculin A), both with cultured primary human T lymphocytes (Fig. 8 A) and K562 transfectants expressing wild-type αLβ2 (Fig. 8 B). By contrast, XVA143 effectively blocked adhesion stimulated by Mn2+ and PMA, but did not inhibit adhesion stimulated by cytochalasin D or latrunculin A (Fig. 8, A and B). These results demonstrate that PMA- and Mn2+-stimulated adhesion (but not cytochalasin D– and latrunculin A–stimulated adhesion) requires conformational communication between the αL I- and β2 I-like domains, and that in all cases, the αL I domain must transit out of the closed, low affinity conformation to support adhesion.

Bottom Line: Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration.Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand.Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

Show MeSH
Related in: MedlinePlus