Absence of specific cell-surface binding of tissue plasminogen activator in uterine cells

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Abstract
Tissue plasminogen activator (tPA), an arginine-specific serine protease, is an oestrogen-regulated protein in uterine and breast cancer tissue. It contains a domain which shares homology with epidermal growth factor (EGF). The aim of the present study was to determine whether specific tPA receptors or EGF receptors mediate the binding of tPA to cells and whether tPA possesses intrinsic mitogenic activity. The binding of 125I-labelled tPA to rat uterine and liver membranes was shown to be non-specific and could not be displaced by unlabelled tPA or EGF. Furthermore, acid washing of cell membranes did not unmask specific tPA-binding sites. In contrast, 125I-labelled EGF binding to both rat uterine and liver membranes was displaced in a dose-dependent manner by unlabelled EGF, and Scatchard analysis of the binding data revealed dissociation constant (Kd) values of 2·4 and 0·71 nm respectively. Unlabelled tPA (up to 20 000-fold excess) did not displace 125I-labelled EGF binding to these membranes. A study of the binding of 125I-labelled tPA and 125I-labelled EGF to endometrial carcinoma cells (Ishikawa), cervical carcinoma cells (HOG-1) and vulval carcinoma cells (A431) showed that up to a 100-fold excess of EGF or a 1000-fold excess of tPA did not displace 125I-labelled tPA binding to these cells. In contrast, 125I-labelled EGF binding was displaced by unlabelled EGF (Kd values for Ishikawa and HOG-1 cells were 2·72 and 1·92 nm respectively) but not by unlabelled tPA (1000-fold excess). Treatment of Ishikawa and HOG-1 cells with EGF (20 ng/ml) for up to 6 days stimulated [3H]thymidine incorporation 1·3- to 2-fold and 3·5- to 4·7-fold respectively. Human recombinant tPA (10 IU/ml) was without effect on both cell lines over 6 days of treatment. It was concluded that although tPA contains an EGF-like domain it does not bind specifically to membrane preparations from rat uterine or liver cells or to cultured uterine cancer cells, and does not displace bound 125I-labelled EGF. Furthermore, tPA does not have mitogenic properties similar to those of EGF.