Abstract
To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.
Highlights
The 2 integrin lymphocyte function-associated antigen-1 (LFA-1)1 (CD11a/CD18 or ␣L2) is a leukocyte-specific adhesion receptor that coordinates different adhesive and signaling interactions within the immune system [1,2,3,4]
Active Regulation of LFA-1 Ligand Binding by Disconnecting the Actin Cytoskeleton Network—LFA-1 expressed on resting peripheral blood lymphocytes is inactive and poorly binds intercellular adhesion molecule-1 (ICAM-1), even when the cells are stimulated by PMA (Fig. 1)
1) Deletion of the 2 and/or ␣L cytoplasmic tail including the KVGFFKR sequence leads to strong ICAM-1 binding by reducing cytoskeleton restraints, enabling the formation of LFA-1 clusters without the necessity of affinity alterations
Summary
The 2 integrin lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18 or ␣L2) is a leukocyte-specific adhesion receptor that coordinates different adhesive and signaling interactions within the immune system [1,2,3,4]. Sites they have to circulate as nonadherent cells This dynamic control of adhesion is regulated by binding strength, and the kinetics of interactions between adhesive ligands and 2 integrins. Activation of LFA-1 likely results in a conformational change in the ␣/ heterodimer, as evidenced by the expression of neoepitopes or activation epitopes (L16 and M24) [6, 23] This has led to the speculation that affinity changes in LFA-1 are associated with conformational alterations, leading to an enhanced binding to its ligand ICAM-1 [24]. Both affinity (active conformation) and avidity (clustering) changes have been considered to be important for strong LFA-1-mediated cell binding [25]. We observed that the cytoplasmic tail of both the 2 chain and the GFFKR sequence in the ␣L cytoplasmic tail play a pivotal role in regulating ligand binding through induction of avidity changes rather than by affinity changes
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