Functional determinants of Rin1 mutants required for their inhibitory activity on endocytosis

Epidermal growth factor receptor, upon ligand binding, induces a series of intracellular events that regulate endocytosis and stimulate different signal transduction pathways. Ras Interference 1 (Rinl) is a multifunctional protein that becomes associated with the activated EGF-receptor and alters both internalization and signaling of the receptor. In this study, a total of ten substitute mutations were created, and their effects on Rinl function were examined. Four of the ten mutants were defective in Rab5 binding, while the other six Rinl mutants partially interacted with Rab5. To study the biological function of Rinl, the effect of Rinl mutants was studied using various biochemical assays including their ability to activate Rab5, to form enlarged early endosomes, and to stimulate endosome fusion. Taken together the data suggest that mutations in the GEF domain of Rinl lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rinl, and also suggest a possible molecular mechanism by which Rinl regulates early endosome fusion