Identification of a binding site in protease nexin I (PN1) required for the receptor mediated internalization of PN1-thrombin complexes.
An overlapping synthetic peptide library was constructed representing most of the mature protease nexin I (PN1) sequence from the amino terminus to the reactive center. This library, along with peptides from the heparin binding domain and from the region carboxyl-terminal to the P1 residue of the cleavage site, was screened for the inhibition of 125I-thrombin (Th)-PN1 complex binding and degradation. A peptide corresponding to residues Pro47-Ile58 in the PN1 sequence was identified as a potent inhibitor of 125I-Th-PN1 complex degradation, although it did not affect binding significantly. Pro47-Ile58 was shown to competitively inhibit the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin receptor-mediated endocytosis of 125I-Th-PN1 complexes in mouse embryo fibroblasts. Pro47-Ile58 is an apparent transition sequence in PN1, separating sheet-6B and helix-B. The sequence of Pro47-Ile58, PHDNIVISPHGI, suggests that it forms a loop structure defined by the seven underlined amino acids bordered by proline residues at each end. These studies are the first to identify a putative binding site in a serine protease inhibitor that is required for LRP-mediated internalization.
Knauer
M F
MF
Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA.
Hawley
S B
SB
Knauer
D J
DJ
D016564:Amyloid beta-Protein Precursor; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D058255:Protease Nexins; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D013917:Thrombin