We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.
CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.
Fc receptors on phagocytic cells in the blood mediate binding and clearance of immune complexes, phagocytosis of antibody-opsonized microorganisms, and potently trigger effector functions, including superoxide anion production and antibody-dependent cellular cytotoxicity. The Fc receptor type III (Fc gamma R III, CD 16), present in 135,000 sites per cell 1 on neutrophils and accounting for most of FcR in blood, unexpectedly has a phosphatidylinositol glycan (PIG) membrane anchor. Deficiency of Fc gamma R III is observed in paroxysmal nocturnal haemoglobinuria (PNH), an acquired abnormality of haematopoietic cells affecting PIG tail biosynthesis or attachment, and is probably responsible for circulating immune complexes and susceptibility to bacterial infections associated with this disease. Although a growing number of eukaryotic cell-surface proteins with PIG-tails are being described, none has thus far been implicated in receptor-mediated endocytosis or in triggering of cell-mediated killing. Our findings on the Fc gamma R III raise the question of how a PIG-tailed protein important in immune complex clearance in vivo and in antibody-dependent killing mediates ligand internalization and cytotoxicity. Together with our results, previous functional studies on Fc gamma R III and Fc gamma R II suggest that these two receptors may cooperate and that the type of membrane anchor is an important mechanism whereby the functional capacity of surface receptors can be regulated.
The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus. This type of linkage has been identified on membrane proteins involved in adhesion and transmembrane signalling and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3.
Granulocyte and natural killer (NK) cell Fc receptors for immunoglobulin G (CD16) differ in only a few amino acids, yet have phosphatidylinositol glycan (PIG) or polypeptide membrane anchors, respectively. Mutagenesis shows that anchoring is regulated by a serine residue near the PIG anchor attachment site in the extracellular domain. The NK cell isoform was not expressed on the surface of COS cells unless cotransfected with a subunit that was expressed in NK cells and that was identical to the gamma subunit of the high affinity IgE Fc receptor (Fc epsilon RI). However, the CD16 sequence and not expression of the gamma subunit is dominant in regulating PIG reanchoring.
To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.
Just as interactions of soluble proteins are affected by the solvent, membrane protein binding is influenced by the surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and kinetic rates of an adhesion receptor (CD16b) placed in three distinct environments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and K562 cells. Effective affinity reflects both the intrinsic receptor-ligand kinetics and the effectiveness of their presentation by the host membranes. Expression of CD16b, a low affinity Fc␥ receptor, was established by either transfection or spontaneous insertion via its glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipette method, indicated a 50-fold increase in effective affinity for receptors on RBCs over CHO and K562 cells, whereas the off rates were similar for all three. Electron microscopy confirmed that specific tight contacts were broad in RBC-RBC conjugates but sparse in CHO-RBC conjugates. We suggest that through modulation of surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.Cell-cell bond formation must be preceded by the creation of a contact zone, a region in which surface-bound receptors and ligands are able to bridge narrow gaps, properly aligning by lateral and rotational diffusion (1). Rough cells initially may form isolated point contacts that over time might be broadened and connected through active membrane processes (2). Alternatively, the cells may simply possess inherently smooth surfaces capable of broad initial contacts. A smooth membrane will not enhance binding to a soluble ligand because the soluble protein can diffuse freely into the membrane folds of rough cells. However, in adhering to immobilized ligand, the ability to form an expansive tight contact area is a distinct advantage. Therefore, cell-cell adhesion depends not only on the intrinsic kinetic rates of the receptor-ligand interaction but also on how effectively the host membranes present these molecules (3).In this study we quantified the effects of various microtopological presentations on cell adhesion by comparing the effective affinity of the same receptor (CD16b) in three distinct host environments: erythrocytes (RBCs), 1 Chinese hamster ovary (CHO) cells, and erythroleukemic K562 cells. Although all other Fc receptors have intracellular domains, CD16b (Fc␥RIIIb) terminates at the lipid bilayer with a glycosylphosphatidylinositol (GPI) moiety and so has no direct cytoskeletal interface. Currently there are no data to suggest the existence of multiple activation states in CD16b. These simplifications permit a more focused investigation into the role of the membrane environment in the functionality of this rec...
Kinetic rates and affinity are essential determinants for biological processes that involve receptor-ligand binding. By using a micropipette method, we measured the kinetics of human Fc␥ receptor III (CD16) interacting with IgG when the two molecules were bound to apposing cellular membranes. CD16 is one of only four eukaryotic receptors known to exist natively in both the transmembrane (TM, CD16a) and glycosylphosphatidylinositol (GPI, CD16b) isoforms. The biological significance of this anchor isoform coexistence is not clear. Here we showed that the anchor influenced kinetic rates; compared with CD16a-TM, CD16a-GPI bound faster and with higher affinities to human and rabbit IgGs but slower and with lower affinity to murine IgG2a. The same differential affinity patterns were observed using soluble IgG ligands. A monoclonal antibody bound CD16a-GPI with higher affinity than CD16a-TM, whereas another monoclonal antibody reacted strongly with CD16a-TM but weakly with CD16a-GPI. No major differential glycosylation between the two CD16a isoforms was detected by SDS-polyacrylamide gel electrophoresis analysis. We suggest a conformational difference as the mechanism underlying the observed anchor effect, as it cannot be explained by the differing diffusivity, flexibility, orientation, height, distribution, or clustering of the two molecules on the cell membrane. These data demonstrate that a covalent modification of an Ig superfamily receptor at the carboxyl terminus of the ectodomain can have an impact on ligand binding kinetics.
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