We present evidence for a discrete 34,000-Da polypeptide with mitogenic activity, associated with plasma membranes from human A431 carcinoma cells. Plasma The role of hormones and growth factors in animal cell proliferation is well established, and many aspects of the interaction of these factors with responsive cells have been worked out (1-5). However, growth regulation by cell-cell contact is poorly understood. It is believed that surface contact between cells is partly responsible for density-dependent inhibition of growth (6, 7). Again, under certain circumstances surface contact can be stimulatory (for example, contact with axonal membranes is known to stimulate Schwann cell division) (8). Thus, cell-cell interaction may generate either mitogenic or antimitogenic signals. In no case has the molecular basis for these growth regulatory effects been identified.In an earlier communication, we reported the properties of a mitogenic activity associated with mouse hepatic membranes (9, 10). The activity was found to be protease sensitive and heat labile. We now find that the plasma membrane from A431 cells, a human epidermoid carcinoma line, is an unusually potent mitogen for target fibroblastic cells. Approximately 30% of the membrane-associated activity can be solubilized with high salt concentration in the absence of detergents, and this apparently represents peripherally attached membrane components. Incubation of this fraction with target fibroblast cells results in selective binding of a single protein of 34,000 Da.These fortunate characteristics-the high mitogenic potency of A431 membranes, the salt extractability of a fraction of this activity, and the selective binding of a particular protein from this fraction by target cells-made possible the studies described here. Evidence so far obtained indicates that the 34,000-Da polypeptide represents a peripherally attached cell-surface mitogen.
MATERIALS AND METHODSCell Culture and Plasma Membranes. Monolayer cultures of Swiss mouse NR-6 cells (11,12) and human A431 cells were grown in Dulbecco's modified Eagle's (DME) medium containing 7% fetal bovine serum and gentamycin at 10 .tg/ml. Membranes were prepared from A431 cells using the procedure of Thom et al. (13).Extract of A431 Membranes. Washed A431 plasma membrane (10 mg of protein) was suspended in 8 ml of 0.7 M NaCl/10 mM Hepes, pH 7.4, and stirred at 40C for 2 hr. The suspension was centrifuged at 100,000 x g for 60 min, and the supernate was concentrated by Amicon ultrafiltration using PM-10 filters. During ultrafiltration, the NaCl concentration was adjusted to 0.15 M.Acetic Acid Treatment of Extract. To extract of A431 membranes treated with a high concentration of salt (1 mg of protein per ml of 0.15 M NaCl/10 mM Hepes, pH 7.4), acetic acid was added to a final concentration of 0.34 M. After incubation at 4°C for 30 min, the mixture was neutralized with saturated Tris solution, and clarified by centrifugation at 100,000 X g for 60 min.Incorporation of [3H]Thymidine into Acid-Insoluble Mat...