Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells.
The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.
Mahdi
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F
Hematology and Oncology Division, Department of Internal Medicine and Pathology, University of Michigan, Ann Arbor, MI 48109-0640, USA.
Shariat-Madar
Z
Z
Todd
R F
RF
Figueroa
C D
CD
Schmaier
A H
AH
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018960:Hyaluronan Receptors; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D007633:Keratins / Q000276:immunology / Q000378:metabolism*; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D011288:Prekallikrein / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D011951:Receptors, Complement / Q000276:immunology / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000032:analysis
C087299:C1QBP protein, human; D002352:Carrier Proteins; D000939:Epitopes; D018960:Hyaluronan Receptors; D007106:Immune Sera; D019679:Kininogen, High-Molecular-Weight; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D055293:Receptors, Urokinase Plasminogen Activator; C032402:complement 1q receptor; D014841:von Willebrand Factor; D007633:Keratins; D011288:Prekallikrein