Integrin Leukocyte Function-associated Antigen-1-mediated Cell Binding Can Be Activated by Clustering of Membrane Rafts

Abstract

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to colocalize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg 2؉ /EGTA unimpaired. In contrast to activation with Mg 2؉ /EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides

“…One of these new proteins, CD18, is the beta-subunit of many heterodimeric leukocyte integrins and is required for numerous interactions of leukocytes with extracellular matrix and cells, including endothelial cells (23). The functional significance of metalloprotease-induced shedding of CD18 is unknown, although previous observations suggest that this event may be responsible for some of the PMA-induced increase in membrane mobility (24,25). The second protein, TEM7-R, was first described as a gene that was up-regulated in malignant colorectal endothelium compared with normal cells (26,27).…”

A Single Amino Acid in the Cytoplasmic Domain of the β2 Integrin Lymphocyte Function-associated Antigen-1 Regulates Avidity-dependent Inside-out Signaling

“…Much attention has been recently given to specialized lipidic membrane microdomains, also termed "rafts." They function as platforms for signaling molecules and are involved in the regulation of LFA-1 function and adhesion through avidity changes (15,30). Whether the PMA-responsive LFA-1 mutant is differently organized in rafts compared with wild type LFA-1 remains so far unsolved.…”

“…The phosphorylation level of these threonines after stimulation with PMA is strongly increased upon pretreatment with okadaic acid, which inhibits serine and threonine phosphatases (25). Threonine-phosphorylated CD18 molecules have been shown to associate with the cytoskeleton (28) and play an important role in the formation of stress fibers and specialized microdomains, such as rafts (15,29,30). However, in our system we could not identify any role of threonine phosphorylation and the PMA responsiveness of LFA-1, although the threonines themselves are a prerequisite for the PMA response together with the mutation L732R.…”

Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions

“…In addition to conformational changes, integrin activation may also depend on receptor clustering or redistribution on the cell surface (37,38). Lub et al (36) reported that activation of PBL leads to uncoupling of CD11a/CD18 from the cytoskeleton and subsequent clustering of the CD11a/CD18 receptor on the cell surface, which, in combination with conformational changes that are induced by inside-out signaling, results in enhanced ligand binding.…”