Cell, Vol. 45, 675-684, June 6, 1966, Copyright 0 1986 by Cell Press Autocrine Saturation of Pro-Urokinase Receptors on Human A431 Cells M. Patrizia Stoppelli,’ Carlo Tacchetti,? M. Vittoria Cubellis: Angelo Corti,* Vincent J. Hearing,5 Giovanni Cassani,* Ettore Appella,§ and Francesco Blasi’ l international Institute of Genetics and Biophysics via Marconi, 10 80125 Naples, Italy t Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20892 *Lepetit S. R A. Research Laboratories via Durando, 38 20100 Milan, Italy 5 Laboratory of Cell Biology National Cancer Institute Bethesda, Maryland 20892 Summary Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid car- cinoma cell line. Most of the cell-associated pro-UPA is on the cell surface, as shown by indirect im- munofluorescence and by surface iodination. Pm- UPA is not an integral membrane protein but is bound to a specific surface receptor that is completely satu- rated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by mincubatlng cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete UPA, which then binds to the surface receptor. We propose that the synthesis of UPA and UPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells. Introduction A large body of evidence shows that plasminogen activa- tors (PAS) play an important role in cellular migratory ac- tivities such as fibroblast motility (Ossowski et al., lQ73), trophoblast implantation (Strickland, Reich, and Sher- man, 1978), and tissue involution (Ossowski, Biegel, and Reich, 1979). PAS may cleave specific proteins, such as plasminogen or other proteins present in the extracellular matrix (Keski-oja and Vaheri, 1982), or they may activate specific proteases involved in basement membrane deg- radation (Liotta et al., 1981; Salo et al., 1982; Sheela and Barrett, 1982). A role for PA in regulating cell migration and invasiveness (Reich, 1978) has been proposed on the basis of the generally increased PA activity in several tumors (see review by Mullins and Rohrlich, 1983) and the correlation between PA activity and tumor growth (Myra-y- Lopez, Reich, and Ossowski, 1983; Myra-y-Lopez et al., 1985). PA may also be involved in tumor metastasis, be- cause antibodies to human urokinase PA (UPA) have been shown to have an inhibitory effect on the metastatic activ- ity of a human tumor cell line inoculated in the chorion al- lantoid chamber of the chicken embryo (Ossowski and Reich, 1983). The detection of PA at the cell surface would help sub- stantiate the involvement of this enzyme in the above- mentioned processes because a cell-associated, exter- nally located proteolytic activity would be advantageous to cells that cross the basement membrane to invade neigh- boring tissues or pass into the circulation. Cell fraction- ation studies have demonstrated, at least in some cells, the association of PA (more specifically, UPA) with the particulate cell fraction, possibly the plasma membrane (Quigley, 1976; Jaken and Black, 1979; Solomon et al., 1980; Zisapel et al., 1982; Lemaire et al., 1983). Human and murine alveolar macrophages have been shown to have a cell-associated UPA activity that is insensitive to UPA inhibitors (Chapman, Vavrin, and Hibbs, 1982; Chap- man, Stone, and Vavrin, 1984). Specific UPA receptors have been described recently in human monocytes and monocyte-like cells (Vassalli et al., 1985; Stoppelli et al., 1985). Because of their properties, these receptors might be the binding sites for the cell- surface uPA. Receptor-bound UPA, in fact, is not internal- ized; rather, it confers surface UPA activity on monocytes. The receptor-binding domain of UPA is located in the 17,OOO dalton amino-terminal fragment (ATF), and it is completely independent of the catalytic domain (Stoppelli et al., 1985). The high affinity of the receptor for UPA en- sures slow rates of dissociation and exchange (Stoppelli et al., 1985; Vassalli et al., 1985). The UPA gene codes for a 50,OOO dalton precursor pro- tein (Salerno et al., 1984), 431 amino acids long (Heyneker et al., 1983; Riccio et al., 1985), that is released as an inac- tive, single-chain pro-uPA form (47,000 daltons, 411 amino acids) into the culture medium by several cell lines (Niel- sen et al., 1982; Wun et al., 1982; Perraiuolo et al., 1984; Eaton, Scott, and Baker, 1984). A proteolytic cleavage eventually activates pro-uPA into the active, two-chain form of UPA (410 amino acids) (GOenzler et al., 1982). Pro- UPA is the major form of UPA in vitro and in vivo (Kielberg et al., 1985). Here we report studies on the localization of pro-uPA in human A431 epidermoid cells. We have found that A431 cells have a surface-associated pro-uPA; pro-uPA is bound to a specific receptor; and pro-uPA is initially secreted and then bound to the receptor. Results Pro-uPA Is Enriched in the Particulate Fraction of A431 Calls Cell fractionation studies have shown that PA in normal and in transformed cells is membrane-associated (Jaken and Black, 1979; Solomon et al., 1980; Zisapel et al., 1982;