Functional determinants of ras interference 1 mutants required for their inhbitory activity on endocytosis

Exp Cell Res. 2009 Mar 10;315(5):820-35. doi: 10.1016/j.yexcr.2008.12.003. Epub 2008 Dec 16.


In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay. In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Down-Regulation / genetics
  • Down-Regulation / physiology
  • Endocytosis / genetics*
  • Endosomes / genetics
  • Endosomes / metabolism
  • Epidermal Growth Factor / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Intracellular Signaling Peptides and Proteins / physiology*
  • Membrane Fusion / genetics
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutant Proteins / physiology
  • Protein Binding
  • Protein Structure, Tertiary / genetics
  • Spodoptera
  • Structure-Activity Relationship
  • rab5 GTP-Binding Proteins / metabolism


  • Intracellular Signaling Peptides and Proteins
  • Mutant Proteins
  • RIN1 protein, human
  • Epidermal Growth Factor
  • rab5 GTP-Binding Proteins