The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.The small GTPase Rab family plays pivotal roles in intracellular membrane trafficking. At present, Ͼ60 Rab GTPases have been identified, and they localize to distinct intracellular compartments that organize transport in specific organelles (1-3). The functional state of Rab GTPases depends on structural conformation, which is determined by binding to guanine nucleotides. In the GDP-bound state, Rab forms a cytoplasmic complex with Rab-GDI, 2 which regulates association with cellular components. After dissociation of Rab-GDI by a GDI dissociation factor on donor membranes, Rab replaces GDP with GTP through its interaction with a GEF at target membranes. This causes a conformational change in Rab that allows recruiting of a variety of downstream effectors onto membranes. During or after membrane fusion, a regulatory protein called Rab-GAP enhances the intrinsic GTPase activity of Rab and promotes GTP hydrolysis. Once this has occurred, GDP-bound Rab re-forms the complex with Rab-GDI and dissociates from membranes.Rab5, the most thoroughly characterized member of the Rab family, is localized mainly to early endosomes (4 -6). Rab5 is involved in the homotypic fusion process of early endosomes and in the budding of clathrin-coated vesicles from plasma membranes and transport to early endosomes (7-9). Therefore, Rab5-GEF plays a key role in the regulation of Rab5 function. To...

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