Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes.

Abstract

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes . Neutrophils treated with the nonionic detergent Lubrol FIX, under controlled conditions, yield intact detergent-insoluble ghosts . Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants . All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent .Biochemical analysis of the ghosts shows that <10% of enzyme markers for the soluble and granule fractions remain, and that >90% of total cell phospholipid is removed during detergent extraction . The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein .Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy . In similar experiments, but using '25 1-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts .A major "51-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase . Further ' 25 1-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A.These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed .Since the wide acceptance of a membrane structure based on the fluid mosaic model proposed by Singer and Nicolson (35), it has become apparent that the lateral mobility of some membrane proteins may be constrained or influenced by their interaction, through the lipid bilayer, with components of the cytoplasm (14). This property of certain membrane proteins can be inferred from their behavior in the presence of multivalent ligands (34) and from lateral diffusion data obtained in photobleaching experiments (30,31,36) . In some cases these phenomena can be enhanced or inhibited by treatment of cells with agents that interfere with microtubule or microfilament THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 743-754 © The Rockefeller University Press " 0021-9525/81/09/0743/12 $1 .00 integrity (13,14), thus leading to the predominant view that these structures are somehow involved either directly or indirectly in...

Characterization and cytoskeletal association of a major cell surface glycoprotein, GP 140, in human neutrophils.

“…Concomitant with actin polymerization, the ligandreceptor complex is converted from a fast dissociating (or low affinity) form to a slow dissociating (or high affinity) form (10, 1 1). Several labs have also demonstrated that following ligand binding, the Con A, Fc, FMLP, CR1, and CR3 (C3bi/MO-1) receptors are recruited to the detergent insoluble actin-containing cytoskeleton of PMNs (3,4,(12)(13)(14). The specific association of these receptors with actin filaments is further supported by the fact that cytochalasin D blocks the formation of ligand-receptor complexes (3,12,14).…”

“…Con A-induced activation of human platelets causes the recruitment of glycoprotein IIb/IIIa and doubles the amount of detergent insoluble actin. In pig PMNs, the recruitment of 217, 170, and 147 kD surface glycoproteins to the detergent insoluble cytoskeletal fraction parallels the capping of Con A receptors (12). In PC12 cells exposure to WGA causes occupied nerve growth factor (NGF) receptors to switch from rapid to slow dissociation kinetics (50).…”

“…Several labs have also demonstrated that following ligand binding, the Con A, Fc, FMLP, CR1, and CR3 (C3bi/MO-1) receptors are recruited to the detergent insoluble actin-containing cytoskeleton of PMNs (3,4,(12)(13)(14). The specific association of these receptors with actin filaments is further supported by the fact that cytochalasin D blocks the formation of ligand-receptor complexes (3,12,14). Jack et al (3) have extended these observations by showing that '251-actin binds to Fc and CR3 (C3bi/MO-l) receptors immobilized on Sepharose-Fc/CR3 receptor antibody beads.…”

Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

“…Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPllb and III exist: one that binds directly to endogenous membrane actin and one that does not.Numerous studies have provided largely indirect evidence for transmembrane interactions among surface proteins and receptors of cells and an internal membrane matrix composed of actin and other cytoskeletal proteins (3,5,7,10,14,16,17,19,23,25,27,31,35,40,45). Indeed, a number of studies have shown that the surface topography and lateral mobility of receptors can be modulated by the underlying cytoskeleton and its associated contractile and motile elements (20,28,30,31,36,37,39).…”

Perfluorocarbon attenuates response of concanavalin A-stimulated mononuclear blood cells without altering ligand-receptor interaction

“…ConA is a plant lectin that binds to mannosylated glycoproteins and glycolipids on the surface of a wide range of cell populations. It represents an established tool to induce cell activation and to study cell membrane structure and dynamics such as the mobility and redistribution of membrane protein receptors (30,31,38). Response of stimulated lymphocytes was determined by means of characteristic IL-2 release.…”