Abstract. Many adhesion receptors have high threedimensional dissociation constants (Kd) for counterreceptors compared to the Kds of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase/Ca (16 txM at 37°C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of ~15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Ka for the CD2/LFA-3 interaction was 21 molecules/txm 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) Kas suggest that cell-ceU contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Ka and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.

. We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion . Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3) . The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2 . Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform . Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the C ELL-CELL interactions are involved in a diverse array of biological functions ranging from morphogenesis to the generation of immune responses . Specific cell surface glycoproteins are known to mediate cell adhesion by ligation with specific counter-receptors. Binding of adhesion molecules allows close apposition oftwo or more cells, during which time the same adhesion molecules or other receptors in the contact area may mediate responses such as signal transduction and cell locomotion (reviewed by Springer, 1990) .Bond formation between two adhesion molecules requires that the cell membranes containing these molecules come into close contact . The frequency of random collision of receptor and counter-receptor molecules is likely to be low during the initial cell-cell contact given the low densities of adhesion molecules on the cell surfaces. A substantial number of adhesive bonds may be required to establish a stable cell-cell interaction, depending on the affinity between the receptor and counter-receptor. It has been shown previously that Fc, receptors can redistribute to the contact area at which the specific ligand is presented on the apposing cell GPI isoform at lower site densities (25-50 sites/pmz), showing that the mobility of LFA 3 is important in adhesion strengthening . At higher site densities (1,500 sites/amt) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform . Reduction of CD2 mobility on Jurkat cells at 5°C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30-fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membranebound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation .

Clustering of beta 1-integrins on adherent cells with antibodies or ligands results in increased tyrosine phosphorylation and activation of a novel focal adhesion tyrosine kinase, pp125FAK. The genes encoding pp125FAK have been cloned previously from both chicken and mouse cDNA libraries, and the deduced amino acid sequences are nearly identical (94%). Two synthetic peptides derived from sequences at the carboxyl terminus of chicken pp125FAK were conjugated to ovalbumin to generate rabbit heteroantisera. Human pp125FAK was immunodetected in both T and B lymphocytes with these antisera. A basal state of pp125FAK tyrosine phosphorylation was observed in T and B lymphocytes, and its expression level was in general augmented among human T- and B-cell leukemia/lymphoma lines. Additionally, the full-length sequence of human T-cell pp125FAK (huT-FAK) was derived from a Jurkat T-cell cDNA library. huT-FAK is structurally identical with both mouse and chicken FAK, and shares 95% amino acid identity with chicken pp125FAK and has 97% homology with the mouse sequence. This high degree of evolutionary conservation between species suggests that pp125FAK is likely to have a crucial function in the cell. Expression of the full-length huT-FAK gene in COS cells showed an immunologically indistinct human pp125FAK protein compared with the endogenous primate pp125FAK. Taken together, the data indicate that this structurally conserved human T-cell pp125FAK likely functions in T- and B-cell lineages, and its altered expression in human lymphocyte tumor cell lines may contribute to their transformed phenotype.

Abstract. Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion . In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules . After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glasssupported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lympho-ELL adhesion plays a fundamental role in the development of multicellular organisms, cell motility, and immunological responses (23,29,33) . In this study we used a micromanipulation technique to determine the direct physical contribution of surface adhesion receptors to antigen-independent T lymphocyte adhesion . In particular, we investigated the adhesion of Jurkat human T lymphoma cells to planar bilayers containing lymphocyte functionassociated antigen 3 (LFA-3)' molecules.The interaction of a T lymphocyte with other cells and with extracellular matrices is mediated by an ensemble of surface adhesion molecules . As demonstrated by the strong adhesion of cultured T cells to cells lacking a specific antigen, some T cell adhesion molecules can function independently of the T cell receptor for antigen . Antigen-independent T-lymphocyte adhesion is due to the binding ofCD2 and LFA-1 receptors on the T cell to LFA-3 and intercellular adhesion molecule (ICAM)-1 and -2 counter receptors on the 1. Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol; ICAM, intercellular adhesion molecule ; LFA, lymphocyte function-associated antigen ; TM, transmembrane .