Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH(i) (designated pH(i)), we have measured pH(i) in NIH3T3 and U251MC cells in the presence of the cytotactin/tenascin fusion proteins and intact cytototactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pH(i) lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pH(i) values than control cells. When two fragments were simultaneously presented, one of which alone increased pH(i) and the other of wh ich alone decreased pH(i), the predominant effect was that of lowered pH,. incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pH(i) changes. The activity of protein kinase C appeared to be important for the changes in pH(i) mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pH(i) elicited by the adhesive fusion protei ns and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pH(i), in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pH(i), are correlated with the phenomenon of counteradhesion whereas the ability to increase pH, is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pH(i). (C) 1994 Wiley-Liss, Inc.
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