Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid

Abstract

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.