Abstract
The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.
Highlights
Among the key regulatory proteins that control endocytic transport and recycling are the Rab family of GTP-binding proteins [5,6,7]
EHD1 Associates Directly with Phosphatidylinositol Lipids— To determine whether EHD1 is capable of directly interacting with lipids, we first utilized an assay in which a wide variety of lipids, immobilized on nitrocellulose, were probed with GST as a control or with a GST-EHD1 fusion protein
EHD3 that was incubated with the liposomes precipitated with liposomes containing PtdIns[3,4,5]P3 and PtdIns[3,5]P2, whereas less than 3% precipitated with PtdIns
Summary
EHD1, the best characterized C-terminal EHD protein far, has a well documented role in controlling recycling from the endocytic recycling compartment to the plasma membrane, a function similar to that attributed to Rab11 [31, 33,34,35,36,37,38,39,40,41,42,43]. The Rab effector Rab11-FIP2, which contains three NPF motifs, interacts with EHD1 through NPF-EH domain interactions and plays a role in recycling from the endocytic recycling compartment [33]. We demonstrate that EHD1 is capable of directly interacting with membranes, by preferentially binding to phosphatidylinositols with a phosphate at position 3 via its EH domain. We describe phosphoinositide-binding as a new function for EH domains, which may in part explain the association of EHD proteins with membranes
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