EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

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.The internalization of receptors is a critical process for eukaryotic cells. Receptors can be internalized from the plasma membrane by a variety of well described mechanisms, including via clathrin-coated pits, independently of clathrin, and through caveolae (1). Once internalized, the small vesicles containing the internalized cargo fuse with early endosomes (also known as sorting endosomes), and the receptors are then either sent to late endosomes and on to the lysosomal pathway for degradation or recycled back to the plasma membrane, where they may participate in additional rounds of internalization (2). Receptor recycling occurs either directly from the sorting endosomes in a process known as "fast recycling" or indirectly in a process termed "slow" or "regulated" recycling (3). Slow recycling has been better characterized and traverses a complex series of tubular and vesicular membrane structures that emerge from the microtubule-organizing center and is collectively known as the endocytic recycling compartment (3, 4). Despite advances in recent years, the process of recycling is not as well understood as internalization.Among the key regulatory proteins that control endocytic transport and recycling are the Rab family of GTP-binding proteins (5-7). Over 60 different mammalian Rab proteins have been identified thus far, and their highly regulated GTP binding and subsequent hydrolysis lead to recruitment of a wide array of effector proteins that are important for vesicular transport and fusion processes within the endocytic pathways. For exa...

Eps15 Homology Domain 1-associated Tubules Contain Phosphatidylinositol-4-Phosphate and Phosphatidylinositol-(4,5)-Bisphosphate and Are Required for Efficient Recycling

“…We and others have demonstrated that in vitro, EHD proteins can interact with an array of different phosphoinositides (Blume et al, 2007;Daumke et al, 2007;Naslavsky et al, 2007). Indeed, full-length EHD proteins and EH domains (by sensitive NMR studies) display broad specificity for phosphoinositides.…”

“…Indirect evidence from our laboratory had previously hinted at a potential role for PtdIns(4,5)P 2 in the generation/maintenance of EHD1-associated tubular membranes through activation of Arf6 (Caplan et al, 2002), which leads to PtdIns(4,5)P 2 generation (Honda et al, 1999). These clues were provided from studies showing that either the Arf6 nucleotide status or direct overexpression of the Arf6 GTP-exchange factor (GEF) EFA6 or the Arf6 GTPase-activating protein ACAP1 led to a loss of EHD1-associated tubular membranes (Caplan et al, 2002).We and others have demonstrated that in vitro, EHD proteins can interact with an array of different phosphoinositides (Blume et al, 2007;Daumke et al, 2007;Naslavsky et al, 2007). Indeed, full-length EHD proteins and EH domains (by sensitive NMR studies) display broad specificity for phosphoinositides.…”

EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology

“…In this study, we highlight a previously undescribed role for the Cterminal EHD protein, EHD3, in the regulation of endosome-toGolgi transport. EHD3 would most likely be categorized as being involved in recruitment because: (1) its C-terminal EH domain, present in other EHD family members, is known to bind to proteins containing NPF motifs (reviewed by Naslavsky and Caplan, 2005;Grant and Caplan, 2008); (2) EHD3 is an ATP-binding protein (Daumke et al, 2007;Lee et al, 2005;Naslavsky et al, 2006); and (3) EHD3 can bind to lipids and membranes directly (Blume et al, 2007;Naslavsky et al, 2007). Indeed, not only EHD3, but its early endosomal interaction partner, rabenosyn-5, as well as STX13, an endosomal SNARE protein, also control earlyendosome-to-recycling-endosome and endosome-to-Golgi trafficking.…”

αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

“…For example, EHD3 depletion disrupts Shiga toxin delivery from the EE/SE to the Golgi (Naslavsky et al, 2009) and impairs transferrin receptor targeting to the ERC (Naslavsky et al, 2006). While the EHD3 mechanism of action is currently unknown, the EHD ATP-binding G domain is critical for phospholipid binding and membrane recruitment (Blume et al, 2007;Daumke et al, 2007;Naslavsky et al, 2007). Following recruitment to membranes, the EH domain might serve as a nucleation site to recruit auxiliary factors that promote cargo sorting within the EE/SE.…”