VITRONECTIN BINDING TO UROKINASE RECEPTOR IN HUMAN BREAST-CANCER

Functional assembly of the plasminogen-dependent proteolytic system on the cell surface requires multiple interactions involving urokinase ( uPA), urokinase receptor (uPAR), plasminogen activator inhibitors, and other molecules that mediate cell migration and adhesion, We analyzed the in vitro interaction of uPAR-containing particulate cell fraction s with the amino-terminal fragment (ATF) of human urokinase and the ma trix-like form of vitronectin, Binding and cross-linking of I-125-labe led ATF to crude membrane extracts from LB6-19 mouse cells overexpress ing human uPARs in the presence of 25 nM urea-denatured vitronectin le d to the formation of, M-r 137,000, 92,000, and 82,000 covalent comple xes, Immunoprecipitation of the preformed cross-linked I-125-labeled c omplexes with anti-vitronectin, anti-uPA, or anti-uPAR antibodies reve aled that the M-r 82,000 and 92,000 species do contain ATF and vitrone ctin and identified the M-r 137,000 species as a ternary complex forme d by ATF, uPAR, and vitronectin, A similar electrophoretic pattern was displayed by acid-pretreated membranes extracted from MCF-7 breast ca rcinoma or HT1080 fibrosarcoma cell lines, as well as a ductal breast carcinoma specimen; the latter exhibited complex formation at concentr ations of vitronectin lower than 10 nM., Finally, uPAR-vitronectin int eraction was further documented by the decreased reactivity of an anti -uPAR polyclonal antibody to acid-pretreated sections of 10 breast car cinomas that had been preincubated with vitronectin. Our findings high light the ability of uPAR to interact simultaneously with vitronectin and uPA in breast cancer, supporting a dynamic coupling of the molecul ar mechanisms underlying plasminogen-dependent matrix degradation and cell adhesion.