Lipophosphoglycan from Leishmania mexicana promastigotes binds to members of the CR3, p150,95 and LFA-1 family of leukocyte integrins

The abundant surface glycolipid, promastigote lipophosphoglycan (LPG), of Leishmania promastigotes was isolated and reconstituted onto the surface of hydrophobic silica beads. These beads bound to both macrophages and monocytes, suggesting that phagocytes possess a receptor(s) capable of recognizing LPG. LPG beads were unable to bind to macrophages isolated from individuals with a genetic deficiency in the CD18 complex of leukocyte integrins (CR3, p150,95, and LFA-1), suggesting that one or more of these receptors were required for binding of LPG. Individual members of the CD18 complex were depleted from macrophages by plating cells on surfaces coated with anti-receptor mAb. These experiments indicated that CR3 and p150,95 from the CD18 complex, were the predominant mediators of attachment of LPG. The phagocyte receptor CR3 expresses two distinct binding sites, one that binds peptide ligands, such as C3bi, and a second site, that recognizes bacterial LPS. Antibody inhibition experiments and competition binding studies with synthetic peptides and soluble LPG indicated that LPG is recognized by the nonpeptide, or "LPS" binding site on CR3.     

cDNA cloning and complete primary structure of the alpha subunit of a leukocyte adhesion glycoprotein, p150,95.

The leukocyte adhesion receptors, p150,95, Mac-1 and LFA-1 are integral membrane glycoproteins which contain distinct alpha subunits of 180,000-150,000 Mr associated with identical beta subunits of 95,000 Mr in alpha beta complexes. p150,95 alpha subunit tryptic peptides were used to specify oligonucleotide probes and a cDNA clone of 4.7 kb containing the entire coding sequence was isolated from a size-selected myeloid cell cDNA library. The 4.7-kb cDNA clone encodes a signal sequence, an extracellular domain of 1081 amino acids containing 10 potential glycosylation sites, a transmembrane domain of 26 amino acids, and a C-terminal cytoplasmic tail of 29 residues. The extracellular domain contains three tandem homologous repeats of approximately 60 amino acids with putative divalent cation-binding sites, and four weaker repeats which lack such binding sites. The cDNA clone hybridizes with a mRNA of 4.7 kb which is induced during in vitro differentiation of myeloid cell lines. The p150,95 alpha subunit is homologous to the alpha subunits of receptors which recognize the RGD sequence in extracellular matrix components, as has previously been shown for the beta subunits, supporting the concept that receptors involved in both cell-cell and cell-matrix interactions belong to a single gene superfamily termed the integrins. Distinctive features of the p150,95 alpha subunit include an insertion of 126 residues N-terminal to the putative metal binding region and a deletion of the region in which the matrix receptors are proteolytically cleaved during processing.     

The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions

Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.     

Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation

The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies of three kindreds

Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.     

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.     

The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion.

Activated monocytic cells and neutrophils adhere to substrates coated with a wide variety of proteins including albumins, catalase, casein, and various extracellular matrix proteins. This adhesion can be specifically inhibited by antibodies directed to the beta 2 integrin subunit. This adhesion to protein substrates shares some similarities with two known protein-protein recognition systems with little apparent binding specificity, namely, the interactions of heat shock proteins and histocompatibility antigens with denatured proteins or peptides. Cell adhesion and affinity chromatography experiments were performed to test the hypothesis that monocytes and neutrophils adhere to and migrate on protein substrates due to the presence of cell surface receptors that recognize common protein structures such as denatured protein epitopes. Adhesion experiments revealed that activated monocytic cells adhere more rapidly and extensively on substrates coated with denatured protein versus native protein. Both adhesion and migration on such substrates in vitro was dependent on beta 2 integrins since blocking antibodies completely interfered with these cellular responses. Affinity chromatography experiments revealed that the Mac-1 and p150,95 integrins could be isolated from monocyte-differentiated HL-60 cells or neutrophils on a denatured protein-Sepharose column. Much greater yields of the receptors were obtained on a denatured versus native protein Sepharose column. The binding of these receptors was specific in that the LFA-1 beta 2 integrin did not bind to the denatured protein column. These data provide evidence that the adhesion of activated monocytes and neutrophils to many protein substrates in vitro is due to the ability of Mac-1 and p150,95 to directly bind to denatured proteins. A model of leukocyte adhesion and invasion whereby activated leukocytes denature extracellular proteins during diapedesis, making them suitable for recognition by beta 2 integrins, is proposed.     

Relative contribution of the leukocyte molecules Mo1, LFA-1, and p150,95 (LeuM5) in adhesion of granulocytes and monocytes to vascular endothelium is tissue- and stimulus-specific

Adhesion of human monocytes and granulocytes to vascular endothelium plays an important role in migration of these cells to inflammatory sites in tissues. A family of three human leukocyte heterodimeric surface molecules named Mo1, LFA-1, and p150,95 (LeuM5) has been shown to mediate leukocyte adhesion to confluent monolayers of human umbilical vein endothelial cells (HUVE). The relative contribution of each of the three molecules in leukocyte endothelial adhesion was studied using a variety of stimuli. Purified human granulocytes and monocytes were radiolabelled and incubated with HUVE for 45 minutes in a 37 degrees C humidified 5% CO2 incubator in the presence or absence of subunit-specific monoclonal antibodies (MAbs). Adhesion was assessed by quantitation of endothelial cell-associated radioactivity and confirmed by microscopic evaluation. MAbs directed against the alpha subunit of LFA-1 as well as to the beta subunit common to all three antigens significantly inhibited unstimulated monocyte adhesion to HUVE. Small but significant inhibiton was also observed using MAbs directed against Mo1a and p150. Phorbol myristate acetate (PMA)-induced grranulocyte adhesion to HUVE was significantly inhibited by anti-Mo1a and anti-beta, but not by anti-LFA-1a or anti-p150. When HUVE were prestimulated by recombinant IL-1, a different pattern of antigen utilization by granulocytes was observed. MAbs directed against each of the three alpha subunits as well as the common beta subunit all inhibited granulocyte adhesion to HUVE. Furthermore the effect of the three anti-alpha subunit MAbs on granulocyte-HUVE adhesion was additive. These studies show that relative contribution of Mo1, LFA-1, and p150,95 to leukocyte endothelial adhesion varies depending on the cell type and the stimulus used. These studies also reveal a novel role for p150,95 in promoting monocyte and granulocyte adhesion to HUVE.     

The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa

We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.     

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.     

Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events.

The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Regulation of cell adhesion receptors by transforming growth factor-beta. Regulation of vitronectin receptor and LFA-1

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) to regulate the expression of members of the alpha beta 2 and alpha beta 3 families of integrins. TGF-beta 1 elevates the expression of vitronectin receptors (alpha v beta 3 integrin) in all cells examined including WI-38 human lung fibroblasts, 3T3-L1 mouse fibroblasts, and MG-63 human osteogenic sarcoma cells. TGF-beta 1 action increases the level of mRNA and the synthesis of vitronectin receptor subunits with t1/2 o 3-4 h and 6 h, respectively. TGF-beta 1 up-regulates expression of the intercellular adhesion receptor, LFA-1 (alpha L beta 2), in THP-1 human monocytic leukemia cells by increasing the synthesis of alpha L subunit but not beta 2 subunit. The increase in alpha L synthesis and assembly into LFA-1 complexes induced by TGF-beta 1 occurs in parallel with elevated fibronectin receptor synthesis in THP-1 cells. These responses to TGF-beta 1 are lost upon phorbol ester-induced differentiation of THP-1 cells into the macrophage phenotype. The results suggest a role of TGF-beta in the regulation of cell-matrix interactions mediated by vitronectin receptors and cell-cell interactions mediated by LFA-1 in the immune system.     

1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin.

The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.     

CR1/CR2 interactions modulate the functions of the cell surface epidermal growth factor receptor

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.     

Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.     

Insulin stimulates proteolysis of the alpha-subunit, but not the beta-subunit, of its receptor at the cell surface in rat liver.

Insulin receptors in rat liver plasma membranes contain two alpha- and two beta-subunits held together by interchain disulphide bonds ([alpha beta]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of Mr-225,000 alpha beta, Mr-200,000 alpha 1 beta, Mr-165,000 alpha beta 1 and Mr-145,000 alpha 1 beta 1 receptor halves containing intact (alpha, beta) or degraded (alpha 1, beta 1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded alpha-subunits to about one-third of the total population during 2 h of incubation at 23 degrees C. beta-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from livers perfused with insulin at 37 degrees C contained degraded alpha-subunits but only intact beta-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded alpha-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the beta-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions.     

The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling

Plasma membrane urokinase-type plasminogen activator (uPA)-receptor (uPAR) is a GPI-anchored protein that binds with high-affinity and activates the serine protease uPA, thus regulating proteolytic activity at the cell surface. In addition, uPAR is a signaling receptor that often does not require its protease ligand or its proteolytic function.uPAR is highly expressed during tissue reorganization, inflammation, and in virtually all human cancers. Since its discovery, in vitro and in vivo models, as well as retrospective clinical studies have shown that over-expression of components of the uPA/uPAR-system correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of cancer. uPAR regulates the cells-extracellular matrix interactions promoting its degradation and turnover through the plasminogen activation cascade.