Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans.
BACKGROUND
The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions.


RESULTS
Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome.


CONCLUSIONS
Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.
George
Manju
M
Division of Molecular Oncology, Evanston Northwestern Healthcare Research Institute, Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, Illinois, USA. m-george2@northwestern.edu <m-george2@northwestern.edu>
Ying
GuoGuang
G
Rainey
Mark A
MA
Solomon
Aharon
A
Parikh
Pankit T
PT
Gao
Qingshen
Q
Band
Vimla
V
Band
Hamid
H
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D010641:Phenotype; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D014168:Transferrin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D034741:RNA, Small Interfering; D014168:Transferrin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C486205:RME-1 protein, C elegans