Trends in Cell Biology
Volume 21, Issue 2, February 2011, Pages 122-131
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Review
EHD proteins: key conductors of endocytic transport

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Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently less was known about the four mammalian dynamin-like C-terminal Eps15 homology domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field.

Introduction

Internalization of receptors and membranes is an essential process required by all mammalian cells, and is vital for multiple cellular processes including nutrient uptake, the regulation of surface receptors, adhesion molecules and ion channels, and synaptic vesicle retrieval in neurons [1]. Whereas some internalized receptors are fated for degradation, a subset of receptors is returned to the plasma membrane where these proteins can partake in additional rounds of internalization. This process, known as endocytic recycling, takes place as receptors are sorted at the early endosome (EE) and transported either directly to the plasma membrane (fast recycling), or through a transitory organelle (slow recycling) known as the endocytic recycling compartment (ERC) (2, 3 for review). Understanding the molecular regulation of these pathways and elucidating the proteins involved has been a challenging process.

Since the early 1990s the Rab family of small GTP-binding proteins has been characterized as a key group of endocytic regulatory proteins 4, 5. Over sixty Rabs have been identified, and many of them participate in the regulation of endocytic transport steps. Rabs generally function by cycling from a GDP-bound inactive state in the cytoplasm to a GTP-bound active state on the membrane of a specific organelle. In their GTP-bound state, Rab proteins have a higher affinity for their interaction partners, known as effectors. Interactions between Rabs and their effectors have been implicated in the specificity of SNARE-based fusion between vesicles and target organelles, and in promoting vesicular transport, fission and fusion [6]. Almost a decade after the Rab proteins were discovered, another family of endocytic regulatory proteins was identified. Known as the C-terminal Eps15 homology domain (EHD) proteins, all four mammalian family members have been implicated in the regulation of specific endocytic transport steps (reviewed in 7, 8; Figure 1). EHDs have been linked to a number of Rab proteins through their association with mutual effectors 9, 10, 11, suggesting a coordinate role in endocytic regulation and highlighting the significance of EHD proteins in these processes (Figure 2).

In recent years there has been intense interest in EHD proteins and an exponential number of EHD papers have been added to the literature. Advances have come from multiple directions; for example, EHD proteins have now been studied in a variety of new model systems that include mice, fruit flies, and plants in addition to worms (Box 1). EHD proteins also appear to regulate receptors in a broad range of human organs and tissues, further bolstering their physiological significance. In parallel, great strides have been made in furthering our mechanistic understanding of EHDs at the molecular and atomic levels, spurring the elucidation of new binding partners and shedding light on the mode by which EHD proteins interact with them. It is therefore timely to review these advances and to identify the challenges that lie ahead. In this review, we shall summarize the roles of the four EHD proteins and tabulate their known interaction partners. Although the review will focus primarily on mammalian EHDs, we shall also provide information on the known functions of EHD proteins in model systems (Box 1). Another focus of this review is to assess the significant amount of new structural information that has arisen recently, and to analyze the implications of these findings for interactions between EHD proteins and their interaction partners. Overall, this summary of EHD proteins and their functions will provide the reader with a clearer window into the world of EHD proteins.

Section snippets

EHD proteins and the regulation of endocytic transport in mammalian cells

Although researchers are only at an early stage in understanding the relationship between EHD proteins and human disease (Table 1), marked advances have been made in understanding the functions of these four proteins at the cellular level.

Choices, choices: selectivity of EHD proteins for specific NPF-containing partners

The four C-terminal EHD proteins have evolutionarily divergent EH domain sequences from the many EH domain-containing proteins (reviewed in 7, 8), and the vast majority of EHD interactions occur via EH domain-NPF motif binding (Table 2). This section will review what determines the selectivity of EHD proteins for specific NPF-containing interaction partners.

Around the bend: EHD proteins as dynamin-like ATPases and ‘benders of membranes’

Based on sequence homology, EHD proteins were originally predicted to contain a nucleotide-binding site similar to those found in Ras and dynamin family GTPases [13]; this notion has received strong support from several studies demonstrating nucleotide binding 10, 35, 36, culminating in the crystal structure of EHD2 in the presence of a non-hydrolyzable ATP analog [36]. Because of its similarity to GTP-binding proteins it was surprising that EHD1 and its paralogs display a much higher affinity

Concluding remarks

Over the past decade, proteins containing a C-terminal EHD have become known as integral regulators of endocytic trafficking. Each of the four EHD proteins regulates selected transport steps, and the EHDs coordinate their activities with members of the Rab family of GTP-binding proteins through common interactions with Rab effectors. Now that these relationships have been defined, and links between EHD proteins and motor proteins have recently been described 24, 42, an important next step will

Acknowledgements

We thank Dr. Keith Johnson for critical reading of this manuscript. Work in the laboratory of S.C. and N.N. is supported by the National Institutes of Health (grants R01GM074876, R01GM087455), the National Center for Research Resources (P20 RR018759), and the Nebraska Department of Health.

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