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Vol. 19, Issue 9, 3823-3835, September 2008

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The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

Shigeo Hara^*,, Etsuko Kiyokawa, Shun-ichiro Iemura, Tohru Natsume, Thomas Wassmer^||, Peter J. Cullen^||, Hiroshi Hiai^¶, and Michiyuki Matsuda^*,,

*Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; National Institute of Advanced Industrial Science and Technology, Biological Information Research Center, Tokyo 135-0064, Japan; ^||Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom; and ^¶Shiga Medical Center Research Institute, Moriyama, Shiga 524-8524, Japan

Submitted March 26, 2008; Revised June 3, 2008; Accepted June 25, 2008
Monitoring Editor: Adam Linstedt

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

Address correspondence to: Etsuko Kiyokawa (kiyokawa{at}lif.kyoto-u.ac.jp)

Abbreviations used: BAR, Bin/amphiphysin/Rvs; DHR, DOCK homology region; MPR, mannose 6-phosphate receptor; PBS, phosphate-buffered saline; PI(3,5)P₂, phosphatidylinositol 3,5-bisphosphate; PIP₃, phosphatidylinositol 3,4,5-trisphosphate; PX, phox homology; RLC, rupture of a lens cataract; SNX, sorting nexin; TGN, trans-Golgi network; Vps, vacuolar protein sorting.