The exon 3 encoded sequence of the intracellular serine proteinase inhibitor plasminogen activator inhibitor 2 is a protein binding domain.
We have used a combination of biochemical and immunological methods to probe for proteins that interact with the cytoplasmic form of plasminogen activator inhibitor 2 (PAI-2) and to identify the structure in PAI-2 that mediates the binding. By affinity chromatography on immobilized PAI-2, we purified a collection of PAI-2-binding proteins. These proteins bound 125I-labeled PAI-2 in vitro (IC50, approximately 10-100 nM) in a calcium-independent reaction that did not abrogate the proteinase inhibitory function of PAI-2. Annexin I was identified among the eluted proteins, and purified annexins I, II, IV, and V, but not III and VI, possessed 125I-labeled PAI-2 binding activity. Immune precipitation by anti-PAI-2 monoclonal and polyclonal antibodies of metabolically labeled melanoma cells treated with a cleavable cross-linker prior to analysis revealed three prominent proteins with apparent masses of 100, 70, and 50 kDa. We localized the protein binding domain in PAI-2 between amino acid residues 66 and 98, as determined by using a PAI-2 mutant lacking this domain and a synthetic peptide spanning this region. This region of PAI-2 corresponds to exon 3 of the gene sequence thought to be critical for PAI-2 functions.
Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark.
P H
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Jensen
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Jensen
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Laug
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Hager
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Gliemann
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Pepinsky
D017302:Annexins / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D005091:Exons; D006801:Humans; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism
D017302:Annexins; D017396:Plasminogen Activator Inhibitor 2; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors