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

Abstract

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.

RIN1 orchestrates the activation of RAB5 GTPases and ABL tyrosine kinases to determine the fate of EGFR

“…S2), reflecting the contribution of RIN1-RAB5 signaling in early endosome fusion (Galvis et al, 2009b). In addition, the RIN1 E574A mutant moderately prolonged downstream signaling, as judged by ERK phosphorylation (Fig.…”

“…This analysis does not distinguish among RAB5 paralogs, although RIN1 has been shown to preferentially activate RAB5A (Chen et al, 2009). Because RIN1 E574A binds poorly to RAB5 (Galvis et al, 2009b), however, the limiting factor may not be RAB5 itself. These results also reinforce the model that positions RIN1 upstream of RABGEF1 in the activation of RAB5 (Xu et al, 2010).…”

“…2A). RIN1 overexpression increased both resting and EGF-stimulated RAB5(GTP) level but a RIN1 mutant with diminished GEF activity, RIN1 E574A (supplementary material Table S1) (Galvis et al, 2009b;Hu et al, 2008;Xu et al, 2010), reduced baseline and EGF-induced RAB5(GTP) to levels below detection ( Fig. 2A).…”

Rin1 restores host phagocytic activity during invasion by Pseudomonas aeruginosa

“…The cDNAs of Rin1 full-length (Rin1 : FL), Rin1 : N (amino acids 1 to 293), Rin1 : C (amino acids 293 to 783), Rin1 : Vps9 (amino acids 293 to 658) and Rin1 : RA (amino acids 452 to 783) were subcloned into the pMX-puro vector as previously described (Barbieri et al, 2003). The Rin1 : Y561F and Rin1 : R629A point mutations were also subcloned into the pMX-puro vector as previously described (Galvis et al, 2009b). The cDNAs were used in FuGENE 6-mediated transfection of 90 % confluent Plate-E cell monolayers to generate retroviruses encoding either PMX-puro vector only, GFP or Rin1 constructs essentially as previously described (Barbieri et al, 2003).…”

“…3d). To obtain a better understanding about the importance of the Rin1 interaction with Rab5 and Ras during active phagocytosis of live P. aeruginosa, we used Rin1 constructs with a functional mutation in the Rin1 : Vps9 domain (Y561F) or in the Rin1 : RA domain (R629A) in the context of the full-length Rin1 (Kiel et al, 2005;Galvis et al, 2009b). We observed a statistically significant decrease in the internalization of P. aeruginosa (w70 %) as compared with control and Rin1-expressing cells (Fig.…”