Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.     

Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor

In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [(3)H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.     

The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.     

Direct determination of the interleukin-6 binding epitope of the interleukin-6 receptor by NMR spectroscopy

All cytokines belonging to the interleukin-6 (IL-6)-type family of cytokines utilize receptors that have a modular build of several immunoglobulin-like and fibronectin type III-like domains. Characteristic of these receptors is a cytokine receptor homology region consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 first binds to its specific receptor and subsequently to a homodimer of the signal transducer protein gp130. The interleukin-6 receptor consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane proximal fibronectin-like domain accounts for more than 90% of the binding energy to IL-6. Here, the key residues of this fibronectin-like domain involved in the interaction with IL-6 are described. Chemical shift mapping data with 15N-labeled IL-6R-D3 and unlabeled IL-6 coupled with recent structural data clearly reveal the epitope within the IL-6R-D3 responsible for mediating the high affinity interaction with its cognate cytokine.     

Characterization of recombinant human CXCR4 in insect cells: role of extracellular domains and N-glycosylation in ligand binding.

The cDNA of the human CXCR4/fusin was isolated from a human HeLa cell cDNA library by PCR and functionally expressed in Sf9 insect cells. The recombinant receptor was found to bind its natural ligand SDF-1alpha with an affinity comparable to that of the native receptor. Sequence-specific antibodies against each of the four extracellular domains were generated and used to investigate the interactions between the different domains of the receptor and the ligand. Each of the four antibodies was found to be able to inhibit ligand binding. CXCR4 was shown to be a glycoprotein. The role of N-glycosylation of CXCR4 in ligand binding was investigated in the insect cells overexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn-11 and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand binding activity and affinity. The double mutant receptor showed little binding activity. Our data suggest that all of the extracellular domains are involved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding.     

Acute upregulation of interleukin-1 receptor by ligand.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.     

Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line

We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters.     

Regulation of the fibronectin receptor affinity by divalent cations

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.     

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1- expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

Tenascin mediates cell attachment through an RGD-dependent receptor

Tenascin is an extracellular matrix glycoprotein expressed in association with mesenchymal-epithelial interactions during development and in the neovasculature and stroma of undifferentiated tumors. This selective expression of tenascin indicates a specific role in cell matrix interactions. We now show that tenascin can support the adhesion of a variety of cell types, including various human tumor cells, normal fibroblasts, and endothelial cells, all of which can attach to a substrate coated with tenascin. Detailed studies on the mechanism of the tenascin-promoted cell attachment were carried out with the human glioma cell line U251MG. The attachment of these cells and others to tenascin were inhibited specifically by peptides containing the RGD cell attachment signal. Affinity chromatography procedures similar to those that have been used to isolate other adhesion receptors yielded a heterodimeric cell surface protein which bound to a tenascin affinity matrix in an RGD-dependent fashion. One of the subunits of this putative tenascin receptor comigrates with the beta subunit of the fibronectin receptor in SDS-PAGE and cross reacts with antibodies prepared against the fibronectin receptor in immunoblotting. These results identify the tenascin receptor as a member of the fibronectin receptor family within the integrin superfamily of receptors. The cell attachment response on tenascin is distinctly different from that seen on fibronectin, suggesting that cell adhesion and motility may be modulated at those sites where tenascin is expressed in the extracellular matrix.     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.     

Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

The extracellular domains of ErbB3 retain high ligand binding affinity at endosome pH and in the locked conformation

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3.     

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.     

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.     

Extracellular matrix and embryonal morphogenesis: role of fibronectin in cell migration

The neural crest provides a useful paradigm for cell migration and modulations in cell adhesion during morphogenesis. In the present review, we describe the major findings on the role of the extracellular matrix glycoprotein fibronectin and its corresponding integrin receptor in the locomotory behavior of neural crest cells. In vivo, fibronectin is associated with the migratory routes of neural crest cells and, in some cases, it disappears from the environment of the cells as they stop migrating. In vitro, neural crest cells show a great preference for fibronectin substrates as compared to other matrix molecules. Both in vivo and in vitro, neural crest cell migration can be specifically inhibited by antibodies or peptides that interfere with the binding of fibronectin to its integrin receptor. However, the migratory behavior of neural crest cells cannot result solely from the interaction with fibronectin. Thus, neural crest cells exhibit a particular organization of integrin receptors on their surface and develop a cytoskeletal network which differs from that of non-motile cells. These properties are supposed to permit rapid changes in the shape of cells and to favor a transient adhesion to the substratum. Recent findings have established that different forms of fibronectin may occur, which differ by short sequences along the molecule. The functions of most of these sequences are not known, except for 1 of them which carries a binding site for integrin receptors. We have demonstrated that this site is recognized by neural crest cells and plays a crucial role in their displacement. It is therefore possible that the forms of fibronectin carrying this sequence are not evenly distributed in the embryo, thus allowing migrating neural crest cells to orientate in the embryo. Fibronectin would then not only play a permissive role in embryonic cell motility, but have an instructive function in cell behavior.