Abstract
The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.
Highlights
Rab guanosine triphosphatases (GTPases) play pivotal roles in intracellular membrane trafficking
RINL did not interact with amphiphysin II, which associates with RIN2 and RIN3 by their PR domains [6] (Fig. 1B)
Since RIN2 and RIN3 have been shown to function as tetramers composed of anti-parallel linkages of two parallel dimers [7], we investigated whether RINL forms homomultimeric complexes in mammalian cells
Summary
Rab guanosine triphosphatases (GTPases) play pivotal roles in intracellular membrane trafficking. More than 60 members have been identified; they are localized in distinct intracellular compartments and regulate intracellular transport [1,2]. Rab, which is the most thoroughly characterized member of this family, is a key regulator of endocytosis, endosome fusion, and endosome trafficking [3]. Like other Rab GTPases, the transition from an inactive state [guanosine diphosphate (GDP)-Rab5] to an active state (GTP-Rab5) is mediated by guanine nucleotide exchange factors (GEFs). Many Rab GEFs have been identified and extensively analyzed [4]. All share a highly conserved vacuolar protein sorting 9 (VPS9) domain, which is required for bindings to, and nucleotide exchange on, Rab proteins. Rabex-5 is a VPS9 domain-containing protein that shows GEF activity for Rab and Rab GTPases; the VPS9 domain structure of Rabex-5 has been determined by X-ray analysis, and four amino acids (i.e., D, P, Y, and T) have been shown to be critical for its GEF activity [5]
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