RESUMEN
The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Ligandos , Receptores de LDL/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Unión Competitiva/genética , Células CHO , Proteínas del Sistema Complemento/genética , Cricetinae , Reactivos de Enlaces Cruzados/metabolismo , Glutaral/metabolismo , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Unión Proteica , ARN Mensajero/análisis , Receptores de LDL/genética , Serina Endopeptidasas/metabolismo , Serpinas/metabolismo , TransfecciónRESUMEN
Very-low density lipoprotein receptor (VLDLR) belongs to the low-density lipoprotein receptor family of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr-40,000 receptor-associated-protein (RAP), and some serine proteinase/serpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with Mr 105,000 and Mr 130,000, respectively [Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjøller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. & Andreasen, P. A. (1995) J. Biol. Chem. 270, 20,855-20,869]. We now demonstrate that these two forms correspond to forms with the absence (type-II) and presence (type-I) of the O-linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrate that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell-surface-associated serine proteinase/serpin complexes in breast carcinomas.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Serpinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conformación de Carbohidratos , Endocitosis , Epitelio , Exones , Glicosilación , Humanos , Inmunohistoquímica , Neoplasias Mamarias Animales/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Conejos , Receptores de LDL/química , Receptores de LDL/genética , Células Tumorales CultivadasRESUMEN
The amino-terminal fragment of human uPA (ATF; amino acids 1-135), 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 His6 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, IIe28, 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 ATF. Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 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 Val20, 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 uPA/uPAR interaction.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Papio , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Estructura Secundaria de Proteína , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Células CHO , Compuestos Cromogénicos , Cricetinae , Escherichia coli/metabolismo , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
The receptor for urokinase-type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase-type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1-284 of human uPAR) was expressed with an N-terminal histidine-tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (corresponding to amino acids 1-277 of human uPAR), was isolated by ligand (uPA) affinity chromatography. Expression in E. coli led to a nonglycosylated form of uPAR, whereas uPAR produced by CHO cells seemed to be glycosylated to a similar extent as the naturally occurring human form of uPAR (as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Prior to immunization the N-termini of the recombinant uPAR variants were determined by amino acid sequence analysis. Polyclonal antibodies were generated in chickens and purified from egg yolk. The reaction patterns of these antibodies were analyzed by Western blot analyses and flow cytofluorometry.
Asunto(s)
Anticuerpos/inmunología , Receptores de Superficie Celular/genética , Activador de Plasminógeno de Tipo Uroquinasa , Secuencia de Aminoácidos , Animales , Anticuerpos/aislamiento & purificación , Secuencia de Bases , Células CHO , Pollos , Cromatografía de Afinidad , Clonación Molecular , Cricetinae , Escherichia coli , Glicosilación , Humanos , Datos de Secuencia Molecular , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales CultivadasRESUMEN
A recombinant soluble human urokinase receptor comprising amino acids 1-277 was cloned and transfected into CHO cells. The mutant protein (rec-uPAR277), purified from the CHO cell supernatant by affinity chromatography on immobilized urokinase (uPA), in a four-fold excess, completely abolished the binding of FITC-labeled pro-uPA to the human ovarian cancer cell line, OV-MZ-6. This invasive and tumorigenic cancer cell line expresses uPA, its inhibitor PAI-1, and the high-affinity receptor for uPA, uPAR. Rec-uPAR277 significantly reduced the proliferation of OV-MZ-6 cells in a concentration-dependent manner without altering the viability of the cells. Invasion of OV-MZ-6 cells tested in an in vitro Matrigel invasion assay was inhibited by rec-uPAR277 up to 75%. In conclusion, these results demonstrate that rec-uPAR277 can function as a scavenger for uPA in vitro by inhibiting proliferation and invasion of human cancer cells.