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 plasmin ogen activator inhibitor 2 (PAI-2) and to identify the structure in PA I-2 that mediates the binding, By affinity chromatography on immobiliz ed PAI-2, we purified a collection of PAI-2-binding proteins. These pr oteins bound I-125-labeled PAI-2 in vitro (IC50, similar to 10-100 nM) in a calcium-imdependent reaction that did not abrogate the proteinas e inhibitory function of PAI-2. Annexin I was identified among the elu ted proteins, and purified annexins I, II, IV, and V, but not III and VI, possessed I-125-labeled PAI-2 binding activity, Immune precipitati on 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 10 0, 70, and 50 kDa. We localized the protein binding domain in PAI-2 be tween amino acid residues 66 and 98, as determined by using a PAI-2 mu tant lacking this domain and a synthetic peptide spanning this region, This region of PAI-2 corresponds to exon 3 of the gene sequence thoug ht to be critical for PAI-2 functions.