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Vol. 20, Issue 22, 4720-4729, November 15, 2009

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Rabex-5 Is a Rab22 Effector and Mediates a Rab22-Rab5 Signaling Cascade in Endocytosis

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104

Submitted June 8, 2009; Revised September 1, 2009; Accepted September 10, 2009
Monitoring Editor: Adam Linstedt

Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5'-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22–Rabex-5–Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.

Address correspondence to: Guangpu Li (guangpu-li{at}ouhsc.edu)

Abbreviations used: BHK, baby hamster kidney; EET, early endosomal targeting; EGF, epidermal growth factor; FL, full length; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione transferase; HB, helical bundle; RFP, red fluorescent protein; WT, wild type.