Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor.
Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.
Bassel-Duby
R
R
Howard Hughes Medical Institute, Dallas, Texas.
Jiang
N Y
NY
Bittick
T
T
Madison
E
E
McGookey
D
D
Orth
K
K
Shohet
R
R
Sambrook
J
J
Gething
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000235:genetics*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; C524898:PLAUR protein, human; C524900:Plaur protein, rat; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D010959:Tissue Plasminogen Activator