Saturation of tumour cell surface receptors for urokinase-type plasminogen activator by amino-terminal fragment and subsequent effect on reconstituted basement membranes invasion

Kobayashi, Hiroshi; Ohi, Hidekazu; Shinohara, Hiromitsu; Sugimura, Motoi; Fujii, Toshiro; Terao, Toshihiko; Schmitt, Manfred; Goretzki, Lothar; Chucholowski, N.; Jänicke, F.

doi:10.1038/bjc.1993.99

Cited by 41 publications

(17 citation statements)

References 31 publications

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“…Recombinant baculoviruses were constructed so as to express the wild-type polyomavirus VP1 protein and modified VP1 proteins containing inserts in each of the four surface-exposed loops. The uPA-related inserts included uPA , the principal binding determinant for uPAR (2); uPA(1-135), the amino-terminal fragment (ATF) whose affinity for uPAR (50% inhibitory concentration [IC 50 ] ϭ 0.12 nM) approximates that of intact uPA (2,47) and which has been widely used in studies of the uPAuPAR interaction (2,33,61); uPA , comprising the morehydrophilic half of ATF; and a phage display peptide (clone 20, 15 amino acids) with high affinity for uPAR (IC 50 ϭ 0.01 M) (24). Additionally, VP1 containing a FLAG epitope in the HI loop was expressed.…”

Section: Resultsmentioning

confidence: 99%

Formation of Polyomavirus-Like Particles with Different VP1 Molecules That Bind the Urokinase Plasminogen Activator Receptor

Shin

Folk

2003

J Virol

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Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.The polyomavirus virion is composed of 72 pentameric subunits arranged in an icosahedral lattice with an approximate diameter of 50 nm (51). Polyomavirus virus-like particles (PyVLPs) of essentially the same dimensions and shape are formed by the self-assembly of the major capsid protein VP1 (55, 56). The structural framework for VP1 is an antiparallel ␤-sheet with jelly roll topology from which emanate four loops (58-60). The BC, DE, and HI loops closely interact and are located at the outward-facing end of the ␤-sheet; the EF loop, originating from the inward-facing end, is located on a side of the ␤-sheet (59, 60). A shallow groove formed between the BC1 and HI loops binds oligosaccharides terminating in ␣-2,3-linked sialic acid, expressed on many animal cells (8,18,58,60); the EF loop, together with the DE loop, contains motifs for integrin binding, which is important for cell susceptibility at a postattachment level (6). Among different strains of polyomavirus, the four VP1 surface-exposed loops exhibit sequence variability (3,17,36,39,53) and the HI loop has been used predominantly as a site for insertion so as to display on the surface of Py-VLPs the Escherichia coli dihydrofolate reductase (DHFR) (22), protein Z (21), a WW domain peptide (57), and a polyanionic adapter sequence (62).The urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked protein expressed on the apical surfaces of endothelial cells (46) and certain epithelial cells (26) and leukocytes (48) to promote proteolysis (13), cell adhesion (7, 66), migration (5, 15), and chemotaxis (9,14,27,28,42,52). uPAR is expressed by many cancer cells, where it is correlated with metastasis and poor prognosis (10, 29-32, 43, 63), and is a potential target for drug and gene therapy. Also, uPAR is expressed on the apical surface of human airway epithelia and might be used to target delivery of DNAs to correct the genetic defect in individuals with cystic fibrosis (12). Se...

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Section: Resultsmentioning

confidence: 99%

Formation of Polyomavirus-Like Particles with Different VP1 Molecules That Bind the Urokinase Plasminogen Activator Receptor

Shin

Folk

2003

J Virol

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“…As matrigel invasion assays showed (Mohanam et al, 1993 andStahl andMueller, 1994), monoclonal antibodies of uPAR were ecient in impairing the cell's invasive ability. Further evidence of the pivotal role of the uPA/uPAR system in invasive behavior of tumor cells has been provided in in vivo and in vitro models (Cohen et al, 1991;Ossowski, 1992;Kobayashi et al, 1993;Kariko et al, 1993;Crowley et al, 1993;Kook et al, 1994, Stahl andMueller, 1994;. If the cell surface-bound uPA limits the rate of invasion, a reduction in surface uPAR should translate into a measurable reduction in invasiveness.…”

Section: Discussionmentioning

confidence: 99%

In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor

Mohanam

Chintala

et al. 1997

Oncogene

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The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Signi®-cant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were signi®cantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any signi®cant dierence however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.

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“…Other uPAR antagonists which block the interaction of functionally active uPA with the tumor-cell-associated receptor (e.g. uPA-derived peptides [20] or a proteolytically inactive uPA mutant [21]) also inhibited the invasiveness or metastasis of cancer cells in vitro and in vivo.…”

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confidence: 99%

Systematic Mutational Analysis of the Receptor‐Binding Region of the Human Urokinase‐Type Plasminogen Activator

Magdolen

Rettenberger

Koppitz³

et al. 1996

European Journal of Biochemistry

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The amino-terminal fragment of human uPA (ATF; amino acids I-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His, tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATE Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPARbinding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thrl8 by alanine or Asn32 by serine had hardly any effect, replacement of Am22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Va120, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated UPNUPAR interaction.Keywords : urokinase-type plasminogen activator ; urokinase-type-plasminogen-activator receptor ; sitedirected mutagenesis ; tumor invasion; ligand-receptor interaction.The serine protease urokinase-type plasminogen activator (uPA) is synthesized and secreted as a single-chain proenzyme, pro-uPA, by a variety of cells, such as monocytes, fibroblasts, and tumor cells. pro-uPA is activated by limited proteolysis into the enzymatically active high-molecular-mass two-chain form of Correspondence to V. Magdolen, Klinische Forschergruppe (DFG)

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Saturation of tumour cell surface receptors for urokinase-type plasminogen activator by amino-terminal fragment and subsequent effect on reconstituted basement membranes invasion

Cited by 41 publications

References 31 publications

Formation of Polyomavirus-Like Particles with Different VP1 Molecules That Bind the Urokinase Plasminogen Activator Receptor

Formation of Polyomavirus-Like Particles with Different VP1 Molecules That Bind the Urokinase Plasminogen Activator Receptor

In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor

Systematic Mutational Analysis of the Receptor‐Binding Region of the Human Urokinase‐Type Plasminogen Activator

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