STRUCTURAL ANALYSIS OF EHD1

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 phosphatidylinositol lipids 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 have demonstrated that phosphatidylinositol-binding may be a more general property shared by other EH-domains. Our analysis reveals a model by which the side chains of several positively charged residues within a 30 amino acid stretch localized primarily to the second and third helices of the EH-domain mediate a direct interaction with phosphatidylinositols.