Related Experiment Videos
Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton
1Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
Insights
Leukocyte function-associated molecule 1 (LFA-1) adhesion is modulated by its cytoplasmic domains through cytoskeleton interactions, not affinity changes. The beta 2 subunit
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Integrin alpha L beta 2, also known as leukocyte function-associated molecule 1 (LFA-1), mediates leukocyte adhesion.
- The cytoplasmic domains of LFA-1 are known to modulate its adhesiveness.
- Modulation of LFA-1 adhesiveness can occur through affinity changes or postreceptor events.
Purpose of the Study:
- To investigate the role of LFA-1 cytoplasmic domains in postreceptor events.
- To elucidate the mechanisms by which LFA-1 cytoplasmic domains regulate cell adhesion.
- To develop a method for studying integrin-mediated adhesion without altering receptor affinity.
Main Methods:
- Constructed chimeric receptors with extracellular alpha IIIb beta 3 domains and intracellular LFA-1 domains.
- Transfected chimeric cDNA into Chinese hamster ovary cells and confirmed heterodimer formation.
- Utilized deletion variants and mutations to lock receptors in a high-affinity state for comparative analysis.
- Employed immunoprecipitation, flow cytometry, scanning electron microscopy, and immunofluorescence microscopy.
Main Results:
- Chimeric receptors mediated fibrinogen adhesion, modulated by phorbol myristate acetate and cytochalasin D, independent of affinity state.
- Specific mutations and deletions in the beta 2 cytoplasmic domain abolished or reduced adhesion without altering affinity.
- Impaired adhesion correlated with reduced focal adhesion formation and cytoskeleton organization.
- The TTT region within the beta 2 cytoplasmic domain is crucial for postreceptor events.
Conclusions:
- Integrin/cytoskeleton interaction, cytoskeleton organization, and cell spreading are postreceptor events modulating LFA-1 adhesion.
- The cytoplasmic domain of the beta 2 subunit, particularly the TTT region, is essential for these postreceptor events.
- Deletion variants offer a novel approach to study integrin-mediated adhesion by stabilizing high-affinity states without affecting downstream signaling.
Abstract:
The integrin alpha L beta 2 (leukocyte function-associated molecule 1, CD11a/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of alpha L beta 2 have been demonstrated to modulate adhesiveness of alpha L beta 2. Affinity changes of alpha L beta 2 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the alpha L beta 2 cytoplasmic domains in postreceptor events we constructed cDNA encoding chimeric proteins with intracellular alpha L beta 2 domains, which are responsible for alpha L beta 2 specific intracellular interactions, and extracellular alpha IIIb beta 3 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The cDNA 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 alpha L beta 2 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 alpha subunit cytoplasmic domains. Cotransfection of this deletion variant with a beta subunit truncation (beta 3 delta 724) and a triple mutation at 758-760 (TTT to AAA) of beta 2 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 alpha L beta 2 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the beta 2 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.