Binding of human single chain urokinase to Chinese Hamster Ovary cells and cloning of hamster u-PAR.
The plasminogen activator, urokinase (u-PA), interacts with the u-PA receptor (u-PAR) which results in enhanced plasminogen activation on cell surfaces. The u-PAR is comprised of three homologous domains of approximately 90 amino acids, defined by the pattern of disulfide bonds. Domain 1 (amino acids 1-87) binds the ligand. Within this domain, Y57, and a site between residues 47 and 53, have been suggested as ligand contact points. Intradomain interactions also contribute to the interaction of u-PA and u-PAR. The interaction of u-PA with its receptor exhibits some species specificity. Previous studies have shown that human u-PA does not bind to the murine u-PAR and murine u-PA does not recognize human u-PAR. However, human u-PA does interact with bovine cells with high affinity. To further examine the interaction of the human ligand with the u-PAR of a different species, we characterized the binding of human 125I single chain u-PA (scu-PA) to hamster cells. Chinese Hamster Ovary (CHO) cells bound human scu-PA with high affinity and capacity (Kd = 1.13 +/- 0.8 nM; Bmax = 5.45 +/- 0.98 x 10(4) sites/cell). In ligand blotting with human 125I-scu-PA, major bands migrating with apparent Mr's of 74, 49 and 38 kDa were observed. The cDNA of hamster u-PAR was cloned and a single 1.4 kb mRNA species identified in Northern blots of CHO cell RNA. For comparison, we also cloned u-PAR cDNA from human THP-1 cells. Our human sequence was identical to those published for U937 and endothelial cells. These sequences were aligned with the published sequences for the murine, bovine and rat u-PAR's to obtain a consensus sequence for five species. The cysteine residues could be aligned for all species. Y57, which has been suggested as a ligand contact point was also conserved across species. In addition, 5 of the 7 amino acids between amino acids 47 and 53 were conserved in all species. Gly283, the most likely glycosyl-phosphatidyl inositol attachment site, was also conserved in all species. The conservation of these amino acid residues across all five species, attests to their importance in u-PAR function. In addition, the results of our studies suggest that the hamster may be a useful small animal model for studies of human urokinase function.
Fowler
B
B
Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Mackman
N
N
Parmer
R J
RJ
Miles
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010960:Plasminogen Activators / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification; D055293:Receptors, Urokinase Plasminogen Activator; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
C524898:PLAUR protein, human; C524899:Plaur protein, mouse; C524900:Plaur protein, rat; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator