Biomedical subjects

U Reuning

Publications and source records attributed to U Reuning.

22 records · Page 2Linked to original sources

PubMed1993

Molecular cloning of cDNA for the bovine urokinase-type plasminogen activator receptor.

We isolated a full length cDNA clone for bovine u-PAR from a bovine aorta endothelial cell cDNA library and compared the structural properties of this receptor protein to the published human and murine sequences. The bovine u-PAR cDNA clone spans a nucleotide sequence stretch of 1335 bp. The open reading frame contains 330 amino acids with a 20 amino acid long putative signal peptide. The mature protein contains 6 potential N-linked glycosylation sites and a high cysteine content (9%). Bovine u-PAR revealed three homologous internal structural repeats. The NH2-terminal repeat containing the u-PA binding site showed 54% identity to the human and murine NH2-terminal domain, compared to 64% identity between human and mouse u-PAR. Southern blot analysis of genomic DNAs from 9 eukaryotic species suggests that the u-PAR gene is conserved in man, monkey, and cow.

Amino Acid Sequence↗

PubMed1993

Two independent binding sites on monolayers of human endothelial cells are responsible for interaction with coagulation factor VII and factor VIIa.

The interaction of radiolabeled factor VII (FVII) and factor VIIa (FVIIa) with endotoxin-stimulated endothelial cells (EC), known to express tissue factor (TF), and unstimulated EC was studied. FVII/FVIIa binding to EC-monolayers was saturable within 4.5-6 h, reversible, temperature and calcium dependent on both, endotoxin-stimulated and on unstimulated EC. Upon 2 h of incubation on EC, FVII was partially converted to FVIIa in the absence of protease inhibitors. The affinity of this binding was Kd = 45.4 +/- 18.7 nM with a calculated number of binding sites Bmax = 3.75 +/- 0.31 x 10(6) molecules/cell. In addition to unlabeled FVII and FVIIa, other vitamin K-dependent proteins reduced binding of [125I]-FVII/FVIIa to about 60-70%, and this type of common binding site for vitamin K-dependent proteins revealed a Kd = 32.2 +/- 5.6 nM and a Bmax = 3.03 +/- 0.14 x 10(6) molecules/cell. Moreover, in the presence of 1 microM prothrombin to suppress common binding sites, only on endotoxin-stimulated EC additional inhibition of FVII/FVIIa binding was achieved by anti-TF antibodies. The characteristics of the FVII/FVIIa-TF interaction with a Kd = 17.2 +/- 5.2 nM and a Bmax = 342,000 +/- 1,100 binding sites/cell revealed a similar saturation kinetics in radioligand binding and in functional factor X activation within 90-120 min. These data indicate the presence of at least two independent binding sites for FVII/FVIIa on stimulated EC of which about 10% are TF specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1992

Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens.

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.

Animals↗

PubMedDate not supplied

Inhibition of the interaction of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) by synthetic peptides.

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.

Amino Acid Sequence↗