Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

TY - JOUR

T1 - Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

AU - Barnathan, E S

AU - Kuo, Alan

AU - Kariko, K

AU - Rosenfeld, L

AU - Murray, Clare S

AU - Behrendt, N

AU - Rønne, E

AU - Weiner, David M

AU - Henkin, J

AU - Cines, D B

PY - 1990/11/1

Y1 - 1990/11/1

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Blotting, Northern

KW - Carrier Proteins

KW - Endothelium, Vascular

KW - Glycoside Hydrolases

KW - Humans

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction

KW - RNA, Messenger

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Tunicamycin

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

KW - Research Support, U.S. Gov't, P.H.S.

M3 - Journal article

C2 - 2171700

SN - 0006-4971

VL - 76

SP - 1795

EP - 1806

JO - Blood

JF - Blood

IS - 9

ER -