Discoidin I-membrane interactions III. Interaction of discoidin I with living Dictyostelium discoideum cells

We have examined the interaction of exogenous ¹²⁵I-discoidin I with living wild type (NC-4) Dictyostelium discoideum cells and the subcellular localization of endogenous discoidin I during development. The living cells bind ¹²⁵I-discoidin I relatively rapidly, with an apparent steady state being reached within 40 min at 23°C. The rate of association is reduced to a similar extent by lowering the temperature to 4°C and by glutaraldehyde fixation of the cells. The level of steady-state binding is not developmentally regulated. Living cells do not degrade significant amounts of the ¹²⁵I-discoidin I during incubations at 23°C. Differentiated cells bind the ¹²⁵I-discoidin I via both the carbohydrate or C site receptors and the ionic or I site receptors, first identified on the corresponding glutaraldehyde-fixed cells (Bartles, J.R. and Frazier, W.A. (1982) Biochim. Biophys. Acta 687, 121-128). Depending on the concentration of ¹²⁵I-discoidin I, 50% or more of the binding to the 8.5-h differentiated cells is via the I sites. Binding exhibits apparent positive cooperativity with respect to discoidin I. This causes the binding to appear nonsaturable, with a capacity for more than 10⁷ discoidin I tetramers per cell. Independent verification of the nonsaturability of the interaction was obtained from experiments using diazotized [¹²⁵I]iodosulfanilic acid to radiolabel the surfaces of cells treated with increasing concentrations of unlabeled discoidin I. The susceptibility of the surface radiolabeled discoidin I to various extractive treatments of the cellular particulate fraction suggests that as much as 10% is very tightly associated with cellular membranes. Dissociation experiments also suggest the existence of an irreversible component of binding, the magnitude of which increases with time of association. While the 8.5-h differentiated NC-4 cells exhibit maximal developmental cohesiveness and contain about 5·10⁶ discoidin I tetramers per cell, cell surface labeling and radioimmunoassay indicate that they display only 1.3·10³ tetramers per cell on their surface and only 6·10³ tetramers per cell in their surrounding extracellular medium.