Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

Abstract

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knockdown of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis

“…Given the role of both EHD1 and EHD3 in regulating primary ciliogenesis, 13,20 it was somewhat unexpected that our previous study in RPE‐1 cells suggested that both remaining EHD paralogs, EHD4 and EHD2, appeared to be dispensable for primary ciliogenesis. Whereas EHD2 is an “outlier” with the least sequence identity and functional homology within the EHD‐family proteins, 37 EHD4 coordinates endosomal fission and recycling through its interactions with EHD1 29,30 and thus we initially chose to more extensively evaluate its potential role in regulating generation of the primary cilium.…”

“…EHD4 hetero-oligomerizes with EHD1 and appears to partially overlap in function with EHD1 and EHD3 in endosomal regulation. 29,30 EHD2, the most disparate EHD protein family member, binds to phosphatidylinositol(4,5)-bisphosphate, localizes to the plasma membrane, [31][32][33] and is involved in caveolae stabilization. 34,35 Despite the crucial roles of both EHD1 and EHD3 in the regulation of primary ciliogenesis, initial studies using the human RPE-1 cells suggested that both EHD2 and EHD4 may be dispensable for mammalian primary ciliogenesis.…”

“…EHD1 and EHD3 belong to a family of four mammalian EHD proteins that display ~65%–86% amino acid identity. EHD4 hetero‐oligomerizes with EHD1 and appears to partially overlap in function with EHD1 and EHD3 in endosomal regulation 29,30 . EHD2, the most disparate EHD protein family member, binds to phosphatidylinositol(4,5)‐bisphosphate, localizes to the plasma membrane, 31–33 and is involved in caveolae stabilization 34,35 .…”

Completing the family of human Eps15 homology domains: Solution structure of the internal Eps15 homology domain of γ‐synergin

“…EH-containing and EH-interacting proteins are often implicated in the regulation of intracellular trafficking as well as cell signaling. 7,21,[26][27][28][29] Known EH domain containing proteins in humans are Eps15, Eps15R, intersectin 1 and 2, Reps1, POB1, Eps15 Homology Domain protein (EHD)1-4 and γ-synergin. Many of the listed proteins have been extensively studied in the past, but less is known about γ-synergin.…”

Cryo-electron tomography reveals structural insights into the membrane binding and remodeling activity of dynamin-like EHDs

“…EHD4/Pincher mediates macropinocytosis required for retrograde endosomal Trk signaling ( 17, 18 ). EHD4 also recruits EHD1 to sorting endosomes via hetero-dimerization ( 19 ). Most recently, a role of an EHD4 complex in the trafficking of vascular endothelial cadherin (VE-cadherin) during angiogenesis was revealed ( 20 ).…”