The hormonal regulation and interaction of concanavalin A and insulin binding in R3230AC mammary adenocarcinoma cells

Abstract

To investigate the effects of concanavalin A on insulin binding to R323AC mammary carcinomas, initial experiments were performed to characterize binding of concanavalin A. Concanavalin A binding was found to be specific and saturable. Equilibrium binding experiments demonstrated that addition of low concentration of concanavalin A enhanced the binding of [ 3H]concanavalin A, suggestive of positively cooperative interactions. Binding of concanavalin A was responsive to hormonal alterations; tumor cells from diabetic rats showed enhanced binding of concanavalin A and insulin compared to cells from intact rats and administration of insulin to diabetic rats returned concanavalin A and insulin binding to levels seen in controls. Incubation of tumor cells with concanavalin A prior to addition of 125I-labelled insulin resulted in a reduction of insulin-binding capacity; succinyl-concanavalin A did not affect binding of insulin. The percent inhibition of insulin binding by concanavalin A was highest at the lower insulin concentrations, providing a linearized Scatchard plot that yielded a calculated K d value comparable to the low-affinity portion of the curvilinear Scatchard plot for insulin binding. The dissociation rate of bound insulin depended on receptor occupancy. Addition of concanavalin A after insulin binding reached equilibrium resulted in increased insulin binding hormone concentrations, decreased rates of dissociation of insulin and a loss of the correlation between receptor occupancy and dissociation rates. Concanavalin A alone demonstrated an insulin-like effect on glucose transport, which in these tumor cells represents a decrease in transport of 3- O-methylglucose. These suggest that binding of both concanavalin A and insulin to cells from this hormonally responsive neoplasm is under insulin regulation and demonstrates similar characteristics to those reported for a variety of normal cells. Furthermore, the interaction between concanavalin A and the cell membranes affects the affinity of the insulin receptor for insulin and appears to decrease the observed negative cooperativity.