Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction.

Abstract

SummaryThe integrin O/L /52 (leukocyte function-associated molecule 1, CDlla/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of O~g /52 have been demonstrated to modulate adhesiveness of at/52-Affinity changes of O/g /52 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the O/g /52 cytoplasmic domains in postreceptor events we constructed eDNA encoding chimeric proteins with intracellular O/g /52 domains, which are responsible for C~L /52 specific intracellular interactions, and extracellular c~iro/33 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The eDNA was stably transfected in Chinese hamster ovary cells and chimeric heterodimer formation proven by immunoprecipitations and flow cytometry. The chimeric receptors mediate adhesion to immobilized fibrinogen, and this adhesion is increased by phorbol myristate acetate and abolished by cytochalasin D. However, neither treatment affects the affinity state of the chimeric receptor, suggesting involvement of the cytoskeleton in the regulation of r /52 mediated cell adhesion. To exclude the possibility of postoccupancy affinity changes of the chimeric receptors, we locked the receptors into a high affinity state by creating a deletion variant. The region deleted (VGFFK) is highly conserved in integrin ce subunit cytoplasmic domains. Cotransfection of this deletion variant with a/3 subunit truncation (/53 a724) and a triple mutation at 758-760 (TTT to AAA) of/32 abolishes adhesion without changing the affinity state. A single mutation (TTT to TAT) reduces adhesion by half without affinity change. Scanning electron microscopy reveals impaired spreading of these truncated/mutated chimeras. Immunofluorescence microscopy demonstrates a correlation between impaired adhesion and a decrease in the ability to form focal adhesions and to organize the cytoskeleton into stress fibers. These results describe the integrin/cytoskeleton interaction, the organization of the cytoskeleton, and cell spreading as postreceptor events modulating c~r/52 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the/52 subunit, and within it the TTT region, are required for these postreceptor events. Additionally, we present a new approach, using deletion variants to lock integrins in a high affinity state without interfering with the investigated integrin/cytoskeleton interaction. This approach may be generally useful to investigate the role of postreceptor events in integrin-mediated cell adhesion and migration.

The Functional Interaction of the β2 Integrin Lymphocyte Function-Associated Antigen-1 with Junctional Adhesion Molecule-A Is Mediated by the I Domain

“…Specific interactions of the ␣ L and ␤ 2 cytoplasmic domains with components of the cytoskeleton (29), such as talin (30), or interactions with specific regulators such as cytohesin-1 (31) appear to be involved in integrin regulation. Similarly, JAM-A expressed at tight junctions of endothelial and epithelial cells interacts via the cytoplasmic PDZ-binding domain with ZO-1 and AF-6 (32), which are both directly linked to the actin cytoskeleton (33,34).…”

Oxidant inhibition of αLβ2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage

“…Further bolstering such a relationship was our finding that DTT promoted rapid reversal of PAO effects on KIM127 epitope expression in parallel with recovery of LFA-1 adhesive function. Interactions between LFA-1 and the cytoskeleton appear to be essential for maintenance of strong adhesion by activated LFA-1 molecules [3,11,12,14,28,54,55]. There is emerging evidence that such interactions may also be involved in maintaining the default low-avidity adhesive state of LFA-1 that is characteristic of resting leukocytes [56].…”

“…Both the ␣L and ␤2 chain cytoplasmic domains contain critical sites, which when deleted or mutated by site-directed mutagenesis result in alteration of LFA-1 adhesive function or response to activating stimuli [10][11][12][13][14]. Phosphorylation events involving protein kinase C (PKC) and serine/threonine phosphatases have been implicated in T and B cell LFA-1 avidity regulation [15][16][17], but the critical site(s) of these enzymatic activities remain to be identified.…”

Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1.

“…In contrast, it has recently been reported that cytochalasins can also induce 132-mediated function (Ross et al, 1992;Elemer and Edgington, 1994;Kucik et al, 1996), whereas others showed no effect from these inhibitors (Diamond and Springer, 1994;Pyszniak et al, 1994). Peter et al (1995) showed that the 12 cytoplasmic domain was involved in the localization of integrins to focal adhesions and in the organization of the actin cytoskeleton into stress fibers (Peter and O'Toole, 1995). Truncation of the cytoplasmic domain of the 2 subunit eliminates LFA-1 binding to ICAM-1, indicating that the cytoplasmic domain of 132 controls adhesiveness (Hibbs et al, 1991a,b).…”

“…Truncation of the cytoplasmic domain of the 2 subunit eliminates LFA-1 binding to ICAM-1, indicating that the cytoplasmic domain of 132 controls adhesiveness (Hibbs et al, 1991a,b). It has been suggested that the reduced adhesiveness of LFA-1 (by deletion of the TTT motif in the cytoplasmic domain of 12) is due to the altered association/organization of the cytoskeleton rather than a change in the affinity of LFA-1 (Peter and O'Toole, 1995). Since the importance of the distribution (clustered/dispersed) of LFA-1 at the cell surface and its attachment to the cytoskeleton became apparent only recently (van Kooyk et al, 1994;Lub et al, 1995), we investigated the role of the cytoskeleton in the activation of LFA-1 when expressed in leukocytes and nonleukocytes.…”