Search PubMed⌕ Search

Biomedical subjects

E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 55 records · Page 3Linked to original sources
PubMed1997

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety.

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate-protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein-protein interaction through the fibronectin type III domains 3-5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein-protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein-protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

Animals↗
PubMed1997

Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF.

Integrin-mediated cell attachment modulates growth responses and growth factors regulate cell attachment. Moreover, both cell attachment to extracellular matrix and mitogenic signaling by growth factors are necessary for the proliferation of most types of normal cells, suggesting that integrin and growth factor receptor signaling pathways meet at some downstream point. We report here that a small, highly tyrosine-phosphorylated fraction of PDGFbeta and insulin receptors co-immunoprecipitates with the alphavbeta3 integrin from cells. The integrin association requires growth factor stimulation of the receptors. Several signaling molecules that are known to be associated with activated growth factor receptors were present in the alphavbeta3 integrin complexes. Mitogenicity and chemotaxis induced by PDGF-BB were enhanced in cells plated on the alphavbeta3 ligand vitronectin compared with cells plated on the beta1 integrin ligand collagen. Thus, the engagement of the alphavbeta3 integrin in cell-matrix interactions appears to coordinate an intense response to growth factors, helping to explain the importance of this integrin for tissue regeneration, angiogenesis and tumor metastasis.

3T3 Cells↗
PubMed1997

Mutation of the protein kinase C phosphorylation site on rat alpha1 Na+,K+-ATPase alters regulation of intracellular Na+ and pH and influences cell shape and adhesiveness.

The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+, K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+, K+-ATPase alpha1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha1 and suggest a connection between this enzyme and cell adhesion.

Animals↗
PubMed1997

Integrins and anoikis.

The loss of integrin-mediated cell-matrix contact induces apoptosis ('anoikis') in certain cell types. Recently it has been shown that protein kinase signaling pathways control anoikis both positively and negatively. Focal adhesion kinase, when activated by integrins, can suppress anoikis. Phosphatidylinositol 3-kinase and the AKT oncoprotein may mediate the anoikis-suppressing effects of focal adhesion kinase. Conversely, the stress-activated protein kinase/Jun amino-terminal kinase pathway promotes anoikis. Latest results indicate that caspase-mediated cleavage of the first component of this latter pathway, MEKK-1, may trigger activation of this pathway in anoikis. In addition, certain integrins may regulate bcl-2 expression levels, possibly adjusting the threshold for anoikis.

Animals↗
PubMed1997

Integrins as signaling molecules and targets for tumor therapy.

Adhesion molecules include ligands and receptors. Together they provide cells with anchorage and traction for migration, and the receptors also mediate signals that control cell polarity, survival, growth, differentiation and gene expression. Integrins are a major group of versatile adhesion receptors that serve both adhesive and signaling functions. They possess shared and unique specifics both outside and inside the cell. Many of the integrins share an affinity toward the RGD recognition sequence in their extracellular matrix ligands, but are still capable of distinguishing different RGD-containing proteins. The shared signaling pathways are likely to include changes in intracellular Ca2+ and PIP2 concentrations, and the activation of protein kinase C and focal adhesion kinase. Examples of integrin-specific signaling include that the alpha v beta 3 integrin (vitronectin receptor) can potentiate the effects of insulin and certain other growth factors and that the alpha 5 beta 1 integrin (fibronectin receptor) supports cell survival in serum-free cultures by up-regulating the anti-apoptosis protein Bcl-2. Another integrin function is that some integrins, in particular alpha 5 beta 1, are necessary for fibronectin matrix formation. Overexpression of alpha 5 beta 1, which results in the assembly of additional fibronectin matrix, reduces tumorigenicity of cultured tumor cells. Systemic treatment of tumor-bearing mice with an artificially generated fibronectin matrix suppresses metastasis. These and other findings indicate that the ligand binding and signaling functions of integrins offer targets for new therapeutic approaches.

Animals↗
PubMed1997

Alpha v integrins as receptors for tumor targeting by circulating ligands.

Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.

Amino Acid Sequence↗
PubMed1996

Betaglycan has multiple binding sites for transforming growth factor-beta 1.

The transforming growth factor-beta (TGF-beta) binding site in betaglycan, the type III TGF-beta receptor, has been variously assigned to the C-terminal half and N-terminal one-third of the extracellular domain. In this study, we show that there are at least two TGF-beta-binding sites in betaglycan. Bacterially expressed fragments bg 1,2 and bg3, which represent the N-terminal two-thirds and C-terminal one-third of betaglycan extracellular domain, both competed for the binding of 125I-TGF-beta to mink lung epithelial cells. 125I-bg1,2 bound to immobilized TGF-beta with an affinity about 4-fold higher than bg3 had. Both bg3 and bg1,2 enhanced the bioactivity of TGF-beta. The whole ectodomain of betaglycan was more active than either bg3 or bg1,2 in the assays. The binding of 125I-bg3 to TGF-beta was inhibited by bg1,2 and vice versa. The binding of 125I-bg3 and 125I-bg1,2 to TGF-beta was also inhibited by the small decorin family of proteoglycans. These results indicate that there are at least two binding sites for TGF-beta in betaglycan and that these sites recognize the same or overlapping sites in TGF-beta.

Animals↗
PubMed1996

Integrin activation by R-ras.

Expression of a constitutively active R-ras converted two cell lines that grow in suspension into highly adherent cells. There was little change in cell surface expression of integrins, but attachment to surfaces coated with the integrin ligands was greatly enhanced. Cells transfected with activated R-ras bound integrin ligands from solution with higher affinities and assembled severalfold more fibronectin matrix than control transfectants. Introduction of a dominant negative R-ras into adherent cells reduced the adhesiveness of the cells, indicating that endogenous R-ras can control the ligand-binding activity of integrins. These results provide a mechanism for the modulation of integrin ligand-binding activity as well as novel function for R-ras.

Animals↗
PubMed1996

Organ targeting in vivo using phage display peptide libraries.

Preferential homing of tumour cells and leukocytes to specific organs indicates that tissues carry unique marker molecules accessible to circulating cells. Organ-selective address molecules on endothelial surfaces have been identified for lymphocyte homing to various lymphoid organs and to tissues undergoing inflammation, and an endothelial marker responsible for tumour homing to the lungs has also been identified. Here we report a new approach to studying organ-selective targeting based on in vivo screening of random peptide sequences. Peptides capable of mediating selective localization of phage to brain and kidney blood vessels were identified, and showed up to 13-fold selectivity for these organs. One of the peptides displayed by the brain-localizing phage was synthesized and shown to specifically inhibit the localization of the homologous phage into the brain. When coated onto glutaraldehyde-fixed red blood cells, the peptide caused selective localization of intravenously injected cells into the brain. These peptide sequences represent the first step towards identifying selective endothelial markers, which may be useful in targeting cells, drugs and genes into selected tissues.

Amino Acid Sequence↗
PubMed1996

Dependence of cyclin E-CDK2 kinase activity on cell anchorage.

Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.

CDC2-CDC28 Kinases↗
PubMed1996

Serglycin-binding proteins in activated macrophages and platelets.

The major proteoglycan in macrophages and platelets is the chondroitin sulphate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulphate-Sepharose was used to isolate proteoglycin-binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein-1 alpha (MIP-1 alpha) precipitated a 14-kDa 35S-methionine-labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of approximately 10 and 14 kDa were precipitated by an antiserum against the murine MIP-1 alpha. Protein sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4-sulphate. Amino-terminal sequencing of two proteins from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PE4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparin sulfate in blocking the binding of [3H] chondrotin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1 alpha, and lysozyme play important role in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.

Animals↗
PubMed1996

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.

Ammonium Sulfate↗