Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors 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 Kd (16 microM at 37 degrees 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 approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 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) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd 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.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3-kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (Y-PO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (Y-PO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y-484 and Y-515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y-405 and Y-445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y-484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y-405 and Y-445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.

To examine the effects of ligand engagement and accessory molecule juxtaposition on T cell receptor (TCR) signaling, we prepared LFA-3/ICAM-1 Rg and LFA-3/VCAM-1 Rg bispecific immunoglobulin fusion proteins (Rg, recombinant globulin). These novel fusion proteins allowed us to examine the effects of ligand driven co-engagement of T cell proteins CD2 and LFA-1 or CD2 and VLA-4 on TCR-dependent mobilization of intracellular Ca2+. We observed that preincubation of resting T cells with LFA-3/ICAM-1 Rg or LFA-3/VCAM-1 Rg fusion proteins resulted in significantly enhanced mobilization of intracellular Ca2+ following TCR-accessory molecule cross-linking relative to T cells preincubated with each of the monospecific Rgs alone or with combinations of the monospecific Rg fusion proteins. In addition, such coengagement stimulated TCR-dependent activation and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1). These results suggest that when T cells interact with antigen presenting cells the engagement of multiple cell adhesion molecules such as CD2, LFA-1, and VLA-4 primes the T cell to respond more effectively to signals delivered through the TCR.

One of the earliest responses of T and B lymphocytes to stimulation through their antigen receptors is the activation of protein tyrosine kinases and the tyrosine phosphorylation of multiple cellular substrates. Here we describe a tyrosine kinase substrate, fakB, a putative homologue of the focal adhesion kinase pp125FAK. Tyrosine phosphorylation of fakB was rapidly augmented in human T and B cells following antigen receptor cross-linking with antibody, while pp125FAK was nonresponsive. Costimulation of the T-cell antigen receptor (TCR/CD3) with either the CD2 or CD4 costimulatory receptors induced synergistic fakB tyrosine phosphorylation in normal human T cells. Engagement of TCR/CD3 induced the stable association of fakB with ZAP-70, the TCR/CD3 sigma-chain-associated tyrosine kinase involved in antigen receptor-induced T-cell activation. In addition, preformed complexes of fakB and ZAP-70 were observed in T-cell leukemia lines. Phosphorylation of fakB on serine, threonine, and tyrosine residues was observed both in vivo and in vitro, where a functional increase of in vitro kinase activity was observed following TCR/CD3 stimulation. fakB is thus a focal adhesion kinase-related tyrosine kinase substrate that is differentially regulated from that of pp125FAK and likely plays a role in antigen-induced lymphocyte signaling.

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.

We herein tested the ability of lymphocytes to utilize the beta1 integrin VLA-4 to mediate cell migration when adhering to its cytokine-inducible endothelial cell ligand VCAM-1 or its extracellular matrix ligand fibronectin. We used an in vitro system consisting of purified VCAM-1/Fc fusion protein or fibronectin immobilized on porous polycarbonate membranes to quantitatively measure the migration efficiency of an Epstein-Barr virus-transformed B cell line (SLA) and T lymphoblasts derived from normal donors. We found that both SLA cells and T lymphoblasts migrated across membranes coated with VCAM-1/Fc or fibronectin in a site density-dependent manner. Above and below an optimal site density of VCAM-1/Fc or fibronectin, the migration efficiency decreased. A 6-20-fold higher number of lymphocytes migrated across membranes coated with VCAM-1/Fc than with fibronectin. The differential migration efficiency is consistent with a higher number of adherent lymphocytes and a higher avidity of adhesion for VCAM-1/Fc than for fibronectin when the ligands were immobilized on plastic, and is independent of the activation state of the cells. These results demonstrated a stringent regulation of migratory response by cell adhesion strength and a delicate balance between stationary and migratory behaviors of a cell on the adhesive substrates. Like the beta2 integrin LFA-1, VLA-4 may be a locomotive adhesion receptor which is involved in the transendothelial migration of lymphocytes and the infiltration of lymphocytes into lymphoid or peripheral tissues by binding to VCAM-1 and fibronectin.

The effect of lengthening the distance in an adhesion molecule between the receptor binding site and the membrane anchor was studied by inserting four Ig-like domains into the two Ig domain lymphocyte function-associated antigen 3 (LFA-3) molecule. The extended molecule expressed in Chinese hamster ovary (CHO) cells bound to CD2 on T lymphocytes 4- to 20-fold more efficiently than the wild-type molecule at 4 degrees C. Treatment of the CHO clones with neuraminidase to remove sialic acid, or with deoxymannojirimycin to reduce the bulk of N-linked glycosylation, showed that adhesion to both the wild-type and the chimeric LFA-3 molecules was under the influence of cell-cell repulsive forces to a similar extent and that these treatments had less effect than lengthening LFA-3. At higher temperatures, such as 22 and 37 degrees C, the efficiency of binding to the wild-type LFA-3 increased to levels comparable with binding to extended LFA-3. Our results suggest that more distal locations of the adhesive binding site from the cell membrane anchor increase the efficiency of cell-cell adhesion by enhancing the frequency of receptor encounter with ligand and that more proximal locations of the adhesive binding site can provide efficient cell-cell adhesion at physiological temperatures.

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 glass-supported 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 lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.

Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidylinositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.

This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton.

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 GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) 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 degrees 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 membrane-bound 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.

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.

The YE1/48 antigen, defined by two rat monoclonal antibodies YE1/48 and YE1/32, is a disulfide-linked dimer of 45- to 50-kDa subunits expressed on murine T cells. It has previously been described as a T cell receptor alpha/beta-like molecule because of its similar m.w., dimeric structure, and isoelectric points comparable to those of the murine T cell receptor. We have now further characterized this antigen, directly compared it with the T cell receptor, and obtained internal amino acid sequences from the purified and trypsin-digested antigen. Endoglycosidase F treatment revealed the peptide cores of the antigen to be approximately 32 to 38kDa under reducing conditions and they possess at least three glycosylation side-chains. On diagonal gel analysis (nonreducing vs reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the YE1/48 antigen and the T cell antigen receptor alpha/beta immunoprecipitated from thymocytes are indistinguishable. However, the two molecules can be distinguished on EL-4 cells by sequential immunoprecipitation using the YE1/48 monoclonal antibody and a rabbit antiserum reactive with the murine T cell receptor. Furthermore, two MBL-2 variant clones, which differ in the level of YE1/48 antigen expression by more than 200-fold, express comparable level of the T cell receptor. Therefore, the YE1/48 antigen and the T cell antigen receptor alpha/beta seem to be different molecules. The YE1/48 antigen was purified from MBL-2(4.1) cells by affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin and the resultant peptides were separated by reverse phase high performance liquid chromatography. The amino acid sequences of several of the YE1/48 tryptic peptides were determined. Upon comparison with the protein sequences in the data base, no identical sequences were detected. These results demonstrated that the YE1/48 antigen is clearly different from the T cell receptor alpha-, beta-, or gamma- chain gene products, and it is a novel T cell antigen not previously described. However, the possibility of homology with other proteins remains undetermined because the tryptic peptides are too short to yield meaningful statistical comparison with the data base.