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.     

Histoplasma capsulatum cyclophilin A mediates attachment to dendritic cell VLA-5

Histoplasma capsulatum (Hc) is a pathogenic fungus that replicates in macrophages (Mphi). In dendritic cells (DC), Hc is killed and fungal Ags are processed and presented to T cells. DC recognize Hc yeasts via the VLA-5 receptor, whereas Mphi recognize yeasts via CD18. To identify ligand(s) on Hc recognized by DC, VLA-5 was used to probe a Far Western blot of a yeast freeze/thaw extract (F/TE) that inhibited Hc binding to DC. VLA-5 recognized a 20-kDa protein, identified as cyclophilin A (CypA), and CypA was present on the surface of Hc yeasts. rCypA inhibited the attachment of Hc to DC, but not to Mphi. Silencing of Hc CypA by RNA interference reduced yeast binding to DC by 65-85%, but had no effect on binding to Mphi. However, F/TE from CypA-silenced yeasts still inhibited binding of wild-type Hc to DC, and F/TE from wild-type yeasts depleted of CypA also inhibited yeast binding to DC. rCypA did not further inhibit the binding of CypA-silenced yeasts to DC. Polystyrene beads coated with rCypA or fibronectin bound to DC and Mphi and to Chinese hamster ovary cells transfected with VLA-5. Binding of rCypA-coated beads, but not fibronectin-coated beads, was inhibited by rCypA. These data demonstrate that CypA serves as a ligand for DC VLA-5, that binding of CypA to VLA-5 is at a site different from FN, and that there is at least one other ligand on the surface of Hc yeasts that mediates binding of Hc to DC.     

Host cyclophilin A mediates HIV-1 attachment to target cells via heparans

The present study proposes a novel mode of action for cyclophilin A (CypA) in the HIV-1 life cycle. We demonstrate that CypA-deficient viruses do not replicate because they fail to attach to target cells. We show that CypA is exposed at the viral membrane and mediates HIV-1 attachment. We identify heparan as the exclusive cellular binding partner for CypA. Furthermore, CypA binds directly to heparan via a domain rich in basic residues similar to known heparin-binding motifs. This interaction between exposed CypA and cell surface heparans represents the initial step of HIV-1 attachment and is a necessary precursor to gp120-binding to CD4. In conclusion, HIV-1 attachment to target cells is a multi-step process that requires an initial CypA-heparan interaction followed by the gp120-CD4 interaction.     

Oligomerization-induced differential dephosphorylation of c-Met receptor tyrosine kinase

Although protein tyrosine phosphatases (PTPs) are significant negative regulators of receptor tyrosine kinase (RTK)-initiated cell signaling, it is unknown how RTK oligomerization modulates the equilibrium established between kinase and phosphatase activity. To determine the impact of oligomerization on the ability of c-MET RTK to undergo dephosphorylation, we examined the relative dephosphorylation kinetics of similarly phosphorylated dimeric TPR-MET and monomeric cytoMET. Notably, we observed that the dephosphorylation kinetics of phosphorylated MET were significantly modulated by its oligomeric state, with the global dephosphorylation rate of TPR-MET severalfold slower than the dephosphorylation rate of monomeric cytoMET. Furthermore, there were important site-specific differences in the dephosphorylation patterns of cytoMET and TPR-MET. Reduced dephosphorylation activity was predicted to eliminate or reduce the requirement of ligand-dependent oligomerization for MET autophosphorylation. This was demonstrated by the rapid phosphorylation of unstimulated c-MET on its activation loop and carboxy-terminal tyrosines following pervanadate treatment of cells expressing c-MET. We conclude that the MET oligomerization state is a critical regulator of its dephosphorylation rate. Thus, oligomerization plays a role in modifying the receptor's kinase and dephosphorylation rates to change the equilibrium levels of phosphorylated and dephosphorylated receptor in response to ligand stimulation, and that this may be a general mechanism utilized by many oligomeric receptor tyrosine kinases for regulation of their activity.     

Platelet thrombospondin mediates attachment and spreading of human melanoma cells

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.     

Progesterone receptor in the chick thymus

Specific, high affinity binding sites for progesterone and promegestone /R-5020/ have been shown to be present in the chick thymus measured by experiments with intact cells or under cell-free conditions. In isolated thymocytes most of the receptor-R-5020 complex is bound to the nucleus. Dissociation constants were determined in thymic cytosol by Scatchard plot analysis and were found to be 3.1 and 2.6 nM for progesterone and R-5020, respectively. On the basis of competition assays the binding sites seemed to be specific for progesterone and R-5020. Glucocorticoids bind only slightly and only at high concentrations. By gel-filtration experiments the thymic R-5020 binding site was shown to be a macromolecule. In vivo treatment of chicks with progesterone or R-5020 caused a significant increase in thymidine kinase activity of the thymus.     

A specific subunit of vitellogenin that mediates receptor binding

Vitellogenin, an estrogen-induced serum protein synthesized in the liver, is composed of two Mr 250K polypeptides. It is specifically transported by a receptor-mediated endocytic process into the developing oocytes of virtually all oviparious animals. Following endocytosis, in the chicken, vitellogenin is specifically processed to yield several smaller products including the phosvitins (PV) and the lipovitellins (LV). These products are then stored within the oocyte until they are degraded during embryogenesis to provide nutrients for the developing embryo. Direct binding studies using iodinated vitellogenin demonstrate that vitellogenin binds to isolated oocyte membranes with a KD of 2.5 microM. Competition studies indicate that PV is a competitive inhibitor of vitellogenin binding. This leads us to propose that the PV portion of the circulating vitellogenin molecule mediates binding and uptake. Direct binding studies using iodinated PV show that PV binds to isolated oocyte membranes with a KD of 2.4 microM. Competition studies also demonstrate that 3.1 microM vitellogenin inhibits 50% of control 125I-PV binding, but IgG and bovine serum albumin at concentrations up to 10 microM have no effect on 125I-PV binding. Another series of competition experiments using a constant amount of vitellogenin and increasing amounts of 125I-PV indicate that vitellogenin acts as a competitive inhibitor of PV binding and has a KI of 2-3 microM. These results support our hypothesis that the receptor which mediates vitellogenin binding and uptake recognizes determinants on the PV portion of the native vitellogenin molecule.     

A ganglioside receptor on lymphocytes mediates recognition of self

Lymphocytes from mouse spleen and thymus form spontaneous rosettes with autologous erythrocytes. Certain gangliosides are described here as potent inhibitors of this cell contact formation. A comparison of several gangliosides shows that lymphocytes from thymus and spleen differ significantly in their reactivity towards GM1, GM3, GD1a, GD1b and GT1b. Differences are also seen between inbred mouse strains.     

A receptor scaffold mediates stimulus-response coupling in bacterial chemotaxis

The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding general features of intracellular signal transduction both in bacterial and eukaryotic cells. Until recently it was thought that the mechanism involved reversible stochastic interactions between dimeric receptors freely diffusing in the cytoplasmic membrane and several soluble signal transduction proteins within the cytoplasm. Recent results have shown that this view is an oversimplification. The receptors and most of the signal transduction proteins are organized together in a higher ordered structure at one pole of the bacterial cell. The scaffolding network within this structure appears to be composed of C-terminal alpha-helical extensions of the membrane chemoreceptor proteins held together in a lattice by tandem SH3-like domains. Results suggest that stimuli are detected through the perturbations they induce in scaffolding architecture.     

Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.     

Progesterone receptor modulators and progesterone antagonists in women's health

Both progesterone receptor modulators (PRMs) as well as pure progesterone antagonists (PAs) have numerous proven and potential therapeutic applications in female health care. Mifepristone, a PRM with only marginal agonistic activity, together with a prostaglandin can terminate pregnancies of less than 9 weeks duration; mifepristone is also used in the preparation of women at later gestational stages whose pregnancies are terminated with prostaglandins or surgery. Mifepristone causes expulsion of the uterine contents following intrauterine fetal death and promotes dilation of the non-pregnant primigravid uterus. It is also effective in the treatment of missed abortion. Together with methotrexate, mifepristone can be used in the medical treatment of ectopic pregnancy. Both PAs and PRMs display antiproliferative effects on the endometrium. Because of this, they have application in the treatment of endometriosis, an estrogen-dependent condition. They may also be utilized to reduce myoma size, acting as both a PA and antiproliferative agent. Unlike GnRH agonists, long-term use in endometriosis and myoma is not associated with loss of bone and hypoestrogenism. PRMs may also be useful in IVF programs to prevent a premature LH surge and to delay the emergence of the implantation window. Some PRMs have potential use as hormone replacement therapy in women during menopause or in those with dysfunctional uterine bleeding.