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Characterization of human endothelial cell urokinase-type plasminogen
activator receptor protein and messenger RNA
ES Barnathan, A Kuo, K Kariko, L Rosenfeld, SC Murray, N Behrendt, E Ronne, D Weiner, J Henkin and DB Cines
Department of Medicine, University of Pennsylvania School of Medicine,
Philadelphia.
Human umbilical vein endothelial cells in culture (HUVEC) express receptors
for urokinase-type plasminogen activators (u-PA). The immunochemical nature
of this receptor and its relationship to u-PA receptors expressed by other
cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead
to an increase in its apparent molecular mass by approximately 40 Kd. The
predominant u-PA binding protein isolated from whole cell detergent
extracts migrated with a molecular mass of approximately 36 Kd using
affinity chromatography. In contrast, when only cell surface proteins were
radiolabeled before extraction, the predominant labeled u-PA binding
protein isolated migrated with a molecular mass of approximately 46 Kd.
Several pieces of evidence suggested that the difference in molecular mass
between these two u-PA binding proteins resulted from glycosylation of a
single receptor protein. First, a polyclonal antibody against u-PA receptor
isolated from phorbol myristate acetate (PMA) stimulated U-937 cells
reacted with both the 36- and 46-Kd proteins on Western blotting. Second,
the size of the unmodified receptor was estimated by amplifying a
full-length cDNA for u-PA receptor from an endothelial cell cDNA library
using the polymerase chain reaction (PCR) and oligonucleotide primers
corresponding to the DNA sequence of the receptor cloned from transformed
human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA
(approximately 1,054 base pairs, bp) and the presence of a single
1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an
unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of
35S-labeled 46-Kd cell surface receptor protein was inhibited when the
cells were grown in the presence of tunicamycin, while the synthesis of the
36-Kd species was unaffected. Moreover, the apparent molecular mass of
purified surface-labeled receptor (approximately 46 Kd) was reduced by
N-glycanase. These studies suggest that the u-PA receptor on the surface of
HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which
is similar immunologically to u-PA receptors on other cell types.
Volume 76,
Issue 9,
pp. 1795-1806,
11/01/1990
Copyright © 1990 by The American Society of Hematology
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