New insights into the tPA-annexin A2 interaction. Is annexin A2 CYS8 the sole requirement for this association?
Annexin A2 has been described as an important receptor for tissue-type plasminogen activator in endothelium and other cell types. Interaction between tissue-type plasminogen activator and its cellular receptor is critical for many of the functions of this protease. The annexin A2 motif that mediates tissue plasminogen activator interaction has been assigned to the hexapeptide LCKLSL in the amino-terminal domain of the protein, and it has been proposed that Cys(8) of this sequence is essential for tPA binding. In an attempt to identify other amino acids critical for tPA-annexin A2 interaction, we have analyzed a set of peptides containing several modifications of the original hexapeptide, including glycine scans, alanine scans, d-amino acid scans, conservative mutations, cysteine blocking, and enantiomer and retroenantiomer sequences. Using a non-radioactive competitive binding assay, we have found that all cysteine-containing peptides, independently of their sequence, compete the interaction between tPA and annexin A2. Cysteine-containing peptides also inhibit tPA binding to the surface of cultured human umbilical vein endothelial cells (HUVEC). Mass spectrometry demonstrates that the peptides bind through a disulfide bond to a cysteine residue of annexin A2, the same mechanism that has been suggested for the inhibition mediated by homocysteine. These data call for a revision of the role of the LCKLSL sequence as the sole annexin A2 structural region required to bind tPA and indicate that further studies are necessary to better define the annexin A2-tPA interaction.
Roda
Oriol
O
Departament de Ciències Experimentales i de la Salut, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra and Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, 08003-Barcelona, Spain.
Valero
M Luz
ML
Peiró
Sandra
S
Andreu
David
D
Real
Francisco X
FX
Navarro
Pilar
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017306:Annexin A2 / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D003545:Cysteine; D004730:Endothelium, Vascular / Q000502:physiology*; D005998:Glycine; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*; D014471:Umbilical Veins
D017306:Annexin A2; D009842:Oligopeptides; D010959:Tissue Plasminogen Activator; D003545:Cysteine; D005998:Glycine