EHD3: A Protein That Resides in Recycling Tubular and Vesicular Membrane Structures and Interacts with EHD1

Abstract

Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22-23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3ƒUTR. One 3.6-kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2-and 3.6-kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule-dependent, membrane tubules. There was a clear colocalization of EHD3-positive structures and transferrincontaining recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N-terminal domain of EHD1 (previously shown to reside in the transferrin-containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N-terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N-terminal or the C-terminal domains, lost their tubular localization. Results of two-hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two-hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule-dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule-dependent movement.

The Endocytic Recycling Protein EHD2 Interacts with Myoferlin to Regulate Myoblast Fusion

“…This result implicates the nucleotide binding state of EHD2 in myoblast fusion. EHD proteins 1-4 contain a central coiled-coil region that mediates oligomerization (39,45). Most recently, the structure of EHD2 was determined where the importance of the nucleotide hydrolysis state was established for its interaction with membrane components (23).…”

Discovery of epigenetically silenced genes in acute myeloid leukemias

“…The protein appears to be a feature of the endocytic recycling compartment (ERC), as it has been shown to colocalize with transferrin containing recycling vesicles. 28,29 A recent study has demonstrated that RNAi-mediated knockdown of EHD3 in HeLa cells results in the failure of internalized transferrin to localize to the ERC, instead remaining contained in large, peripheral organelles. 30 In the same study, the authors note recent evidence implying that blocking vesicle trafficking to the ERC may, in fact, increase the rate of recycling.…”

ATP Binding Regulates Oligomerization and Endosome Association of RME-1 Family Proteins

“…RME-1/mRme-1/EHD1 has been shown to interact with several other proteins that are likely to function with RME-1 during membrane transport processes. EHD1/mRme-1 has been shown to form dimers or hetero-oligomers with the highly related protein EHD3 (14). In addition, EHD1/mRme-1 interacts through its EH domain with an actin-associated protein EHBP1 that, like EHD1/mRme-1 itself, is required for insulinstimulated translocation of the GLUT4 to the plasma membrane (13).…”

“…RME-1 has three predicted domains: an N-terminal P-loop nucleotide-binding domain, a central domain predicted to form a coiled-coil, and a C-terminal eps15 homology (EH) 1 domain (10). Yeast two-hybrid analysis suggested that EHD1 selfdimerizes or oligomerizes possibly through the activity of its coiled-coil domain (14,15). EHD1 also forms hetero-dimers or oligomers with EHD3, another member of the mammalian RME-1 protein family (14).…”

“…Yeast two-hybrid analysis suggested that EHD1 selfdimerizes or oligomerizes possibly through the activity of its coiled-coil domain (14,15). EHD1 also forms hetero-dimers or oligomers with EHD3, another member of the mammalian RME-1 protein family (14).…”