TY - JOUR

T1 - The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

AU - Behrendt, N

AU - Rønne, E

AU - Ploug, M

AU - Petri, T.

AU - Løber, D.

AU - Nielsen, Lars S

AU - Schleuning, W.D.

AU - Blasi, F.

AU - Appella, E

AU - Danø, K

PY - 1990/4/15

Y1 - 1990/4/15

N2 - The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.

AB - The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.

KW - Amino Acid Sequence

KW - Amino Acids

KW - Cell Line

KW - Chromatography, Affinity

KW - Electrophoresis, Polyacrylamide Gel

KW - Enzyme Precursors

KW - Genetic Variation

KW - Glycosylation

KW - Humans

KW - Molecular Sequence Data

KW - Molecular Weight

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Tetradecanoylphorbol Acetate

KW - Urokinase-Type Plasminogen Activator

KW - Journal Article

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

M3 - Journal article

C2 - 2156852

VL - 265

SP - 6453

EP - 6460

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 11

ER -