ABSTRACT
The cDNA probe puPAR-2 detects two PstI polymorphisms in the urokinase-type plasminogen activator receptor gene (PLAUR). This probe and the polymorphic system are described.
Subject(s)
Genes/genetics , Plasminogen Activators/genetics , Polymorphism, Restriction Fragment Length , Receptors, Cell Surface/genetics , Urokinase-Type Plasminogen Activator/genetics , Blotting, Southern , DNA Probes , Humans , Linkage Disequilibrium , Receptors, Urokinase Plasminogen ActivatorABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.
Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 19 , Receptors, Cell Surface/genetics , Urokinase-Type Plasminogen Activator/genetics , Animals , Genetic Linkage , HeLa Cells , Humans , Hybrid Cells , Mice , Receptors, Urokinase Plasminogen ActivatorABSTRACT
We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates transcription of the u-PAR gene in U937 cells, and this is responsible at least in part for the accumulation of the u-PAR mRNA and for the subsequent increase in urokinase-binding capacity.
Subject(s)
Monocytes/drug effects , RNA, Messenger/analysis , Receptors, Cell Surface/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects , Urokinase-Type Plasminogen Activator/metabolism , Blotting, Northern , Cell Line , Cycloheximide/pharmacology , DNA/genetics , Humans , Monocytes/enzymology , Monocytes/metabolism , RNA, Messenger/genetics , Receptors, Cell Surface/drug effects , Receptors, Urokinase Plasminogen ActivatorABSTRACT
The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.
