Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Determination of binding strength and kinetics of binding initiation

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5×10⁻³ dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1–10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.     

Adhesion sites of murine fibroblasts on cold insoluble globulin-adsorbed substrata

The attachment and detachment behavior of three mouse fibroblast cell lines adhering to plastic tissue culture substrata coated with the serum protein cold-insoluble globulin (CIg) resembles that seen on the usual serumcoated substrata. The transformed cell line SVT2 spreads more extensively on the CIg-coated than on the serum-coated substratum, while the nontransformed Balb/c 3T3 line and concanavalin A-selected “revertant” of SVT2 are equally well spread on both substrata. In all three cases, immunofluorescence microscopy using antibodies to CIg suggests that the cells are more tightly apposed to the CIg-coated substratum than to the serum-coated substratum. Substrate-attached material (SAM), which contains cell-substratum adhesion sites and which is left after EGTA-mediated detachment of cells, is enriched for cell surface fibronectin and glycosaminoglycans (GAG). When cells are seeded onto CIg-coated substrata rather than serum-coated substrata, there is an increased deposition of GAG but a comparable deposition of cellular proteins. The protein distribution of the two types of SAM are identical as analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, including fibronectin content. This indicates that substratum-bound CIg cannot functionally substitute for cell surface fibronectin in these adhesion sites. Analysis of the GAG deposited on CIg-coated substrata reveals that hyaluronate and the chondroitins are increased to a much greater extent than heparan sulfate; however, the ratio of hyaluronate to the various chondroitin species is invariant. These data provide further evidence that hyaluronate and the chondroitins are deposited in adhesion sites in well-defined stoichiometric proportions, possibly as supramolecular complexes, and that CIg may mediate adhesion of cells in the serum layer by binding to GAG-containing proteoglycans.     

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.     

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering

The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed <1 s after contact with higher fibronectin surface densities. Cell treatment with microfilament inhibitors or a neutral antiintegrin antibody decreased bond number without changing aforementioned kinetic parameters whereas a function enhancing antibody increased the rate of bond formation and/or the lifetime of intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor association. This interpretation accounts for the functional importance of integrin clustering.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells

Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs) were identified as fibronectin binding proteins (FBPs): OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells). Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

A Mr 72K cell surface concanavalin A binding glycoprotein is specifically involved in the spreading of chick embryo fibroblasts onto laminin substrate

In the present study we have identified a 72-kDa cell surface concanavalin A binding glycoprotein (cbg 72) involved in the chick embryo fibroblast (CEF) adhesion onto laminin (LM) substrate. The cbg 72 was shown to interact specifically with immobilized laminin and to be resistant to Triton X-100 extraction when CEF were plated on laminin substrate but not on fibronectin (FN) substrate. This behavior suggested that cbg 72 could interact with cytoskeletal elements during cell spreading onto LM. This assumption is also in good agreement with the partitioning of cbg 72 in Triton X-114. Isolated cbg 72 specifically inhibited CEF spreading onto LM after their initial attachment, whereas cbg 72 did not impair the spreading of CEF onto FN. These data provide a molecular explanation to the inhibition of CEF spreading onto LM observed in the presence of the lectin concanavalin A (P. Codogno, M.-A. Doyennette-Moyne, J. Botti, and M. Aubery, 1988, J. Cell Physiol. 136, 463-470). Moreover, these results provide evidence for the role of a novel LM binding glycoprotein during the adhesion of mesenchymal derived cells. The relationship between cbg 72 and other known cell surface LM binding sites or receptors is discussed.     

Lectin receptor proximity on HL-60 leukemia cells determined by fluorescence energy transfer using flow cytometry

Fluorescence energy transfer using flow cytometric measurements was utilized to determine the proximity of concanavalin A receptors on the surface of HL-60 promyelocytic leukemia cells before and after induction of differentiation. The HL-60 cells were induced to differentiate into granulocytes using dimethylsulfoxide and into macrophages using 12-O-tetradecanoylphorbol-13-acetate. Concanavalin A was labeled with either fluorescein (donor chromophore) or tetramethylrhodamine (acceptor chromophore), and these species were used to determine lectin proximity. With granulocytic differentiation, the amount of concanavalin A bound remained constant, but a decrease in receptor density was observed. During macrophage differentiation, however, both receptor density and receptor number increased. The increase in concanavalin A binding during differentiation appears to be a result of maturation rather than an initiating event.     

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.