On normal resting lymphocytes, LFA is in an inactive state that poorly binds ICAM.Several recent publications have demonstrated that temporary dislodgment of LFA from the cytoskeleton network facilitates ICAM binding, following TCRCD or PMA activation. This temporary disconnection from the cytoskeleton restraints by cytochalasin D may facilitate redistribution of LFA receptors on the cell membrane altering LFAICAM avidity interaction.In this study, we aimed to investigate the role of the a andbcytoplasmic tails of LFA in regulating ICAM binding through avidity changes or affinity changes.Most Hematoxylin integrins contain the conserved DLRE motif in thebcytoplasmic tail.Deletion of this sequence has already been shown to lead to an active receptor for various integrins, enabling it to bind spontaneously ligand. Comparison of the positions at which the distinctbcytoplasmic tail were truncated, would suggest that deletion of the conserved aspartic acid residue corresponding to position in the b tail results in a constitutively active molecule, indicating that this residue is most important in regulating integrin activation.In contrast, we observed that in deletion mutants in which this conserved aspartic acid residue is not removed, the integrin activity can still be regulated.Also the a cytoplasmic tails of other integrins contain the highly conserved GFFKR sequence that when deleted enhances ligand binding, similar to the DLRE motif. This notion is supported by our observation that only those aL truncation mutants, which did not contain the KVGFFKR Cozymase region, are constitutively active.A, adhesion of cytoplasmic chimeric LFA mutants; B, adhesion of aL deletion mutants; C, adhesion of b deletion mutants.To investigate whether the spontaneous activation of LFA, due to truncation of the cytoplasmic tail, was the result of affinity or avidity alterations, we determined the minimal concentration of soluble ICAM to bind the various LFA transfectants. Because clustering of LFA on these cytoplasmic tail mutants might be due to a reduced capacity to interact with the cytoskeleton, we investigated whether disruption of the actin cytoskeleton by cytochalasin D affected adhesion.As expected, no reduction of spontaneous adhesion to ICAM was observed, indicating that the cytoskeleton is not attached to LFA when thebor a cytoplasmic tails are truncated.Therefore, no postreceptorbinding events that depend on the attachment of cytoskeleton are observed. By contrast, when the entire aL cytoplasmic tail was deleted, LFA was less clustered and was still able to spread on ICAM similar to wildtype LFA.It has been suggested that the altered adhesiveness due to mutation of the threonine triplet is caused by an altered cytoskeletal associationorganization and not to an affinity change in LFA. Clustering of integrins on the cell surface can also colocalize important kinases essential for proper signal transduction. Not only the intracellular conformation or association with regulatory proteins is affected by clustering of integrins on the cell surface, but the extracellular conformation is altered also, as evidenced by enhanced L and M epitope expression when the b or aL cytoplasmic domain was deleted. This may be attributed to distinct interactions with cytoplasmic proteins affecting the extracellular conformations of the integrin molecule.Because KLFA transfectants express ICAM, we investigated whether initial cell contact with ligand during culture may result in the dynamic clustering of integrins thereby augmenting the avidity for ligand.