Interaction of the fibrinolytic receptor, annexin II, with the endothelial cell surface. Essential role of endonexin repeat 2.
Endothelial cells express a cell surface co-receptor for plasminogen and tissue plasminogen activator (t-PA) which we recently identified as annexin II (Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197). This protein enhances the catalytic efficiency of t-PA-dependent plasmin generation by 60-fold (Cesarman, G. M., Guevara, C. A., and Hajjar, K. A. (1994) J. Biol. Chem. 269, 21198-21203). Here, we demonstrate that annexin II is constitutively translocated to the endothelial cell surface within 16 h of biosynthesis, and that cell surface annexin II comprises 4.3 +/- 1.0% of the total cellular pool. Exogenous 125I-annexin II bound to EGTA-washed endothelial cells with high affinity (Kd 49 nM) and in a calcium-dependent (I50 = 3 microM), phospholipid-sensitive manner. Peptides KASMKGLGTDED and YDSMKGKGTRDK, mimicking the calcium-binding "endonexin" motif (KGXGT) of annexin II, blocked its interaction with endothelial cells. Recombinant annexin II, bearing the calcium-binding site substitution D161A of core repeat 2, failed to compete with binding of the wild type protein to the cell surface, while E246A and D321A mutants, corresponding to core repeats 3 and 4, behaved as effective competitors. These data suggest that translocated annexin II interacts with cell surface phospholipid via a high affinity calcium-dependent binding site that includes residues 118-122 (KGLGT) and the coordinating Asp161 of core repeat 2. Thus, calcium-regulated expression of annexin II on the endothelial cell surface may play a central role in control of plasmin-mediated processes.
Hajjar
K A
KA
Department of Pediatrics, Cornell University Medical College, New York, New York 10021, USA.
Guevara
C A
CA
Lev
E
E
Dowling
K
K
Chacko
J
J
D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005342:Fibrinolysis; D006801:Humans; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D011485:Protein Binding
D017306:Annexin A2; D017931:DNA Primers; D010958:Plasminogen; D002118:Calcium