Analysis of Protein-Protein Interactions of Eps15 Using Fluorescence Resonance Energy Transfer (FRET)

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Endocytosis, the process whereby cells internalize regions of their cell surfaces (plasma membranes), is a critical property of living cells. For single-celled organisms, endocytosis is an important mechanism for sampling the environment and for ingesting microbial food. Cells that are part of a multicellular organism must communicate with one another in order to coordinate their activities. Often, such communication is in the form of signaling molecules, such as hormones, that are produced by one cell type and that bind to specific receptors on other (target) cell types. Endocytosis of the receptor-signal complex is generally then a necessary second step in order for the target cell to respond appropriately to the signal. Endocytosis is also the means whereby cell surface components that are no longer needed are removed from the surface. Because of the critical importance of endocytosis to the lives of cells and organisms, the mechanism of endocytosis has been an important focus of cell biological research. This project addresses a very specific molecular aspect of the endocytic machinery of cells.Eps15 is a recently identified component of the cellular internalization machinery. Eps15 is constitutively associated with the a-adaptin subunit of clathrin adaptor protein AP-2, and is located in the plasma membrane clathrin-coated pits and vesicles. The importance of Eps15 in the clathrin-mediated process has been demonstrated in functional studies that used dominant-negative Eps15 mutants and microinjection techniques. However, the precise function of Eps15 in clathrin vesicle formation is unknown. Eps15 is a prototype of the large family of proteins containing the EH (for Eps15 Homology) domain, conserved protein-protein interaction modules that are found in mammals, frog, fly, nematode and yeast proteins. EH domains of Eps15 bind several proteins containing Asp-Pro-Phe (NPF) sequence motifs. The role of EH domain interactions in endocytosis remains to be determined. Eps15 is also constitutively dimerized/tetramerized, although the mechanism of Eps15 oligomerization is not fully understood. Elucidation of the molecular details of Eps15 interactions is essential for understanding the function of this protein.The goal of the project is to analyze the Eps15 interactions that are important for the steps of endocytosis regulated by Eps15. New technical approaches will be developed to study the dynamics of Eps15 complexes in living cells. A high-resolution microscopic technique called Fluorescence Resonance Energy Transfer (FRET) will be used to investigate protein interactions. FRET is based on the transfer of energy between two proximate fluorochromes. Recent development of green fluorescent proteins (GFP) with different spectral characteristics allows utilization of chimeric proteins tagged with such molecules as tools for the analysis of near-neighbor protein interactions in live cells. Dr. Sorkin has already constructed and characterized a GFP-Eps15 chimera for use in a FRET-based assay. Additional chimeric Eps15 constructs tagged with yellow (YFP) or cyan fluorescent protein (CFP) will be constructed and co-expressed in the same cells to study the mechanisms of dimerization and tetramerization of Eps15. To analyze the interactions of Eps15 with cargo receptors and the components of the internalization machinery, Eps15-CFP will be co-expressed with YFP-tagged epidermal growth factor (EGF) receptor, clathrin adaptors, clathrin assembly lymphoid myeloid (CALM) protein or intersectin. In FRET experiments, CFP, when excited by low energy light of an appropriate wavelength, can transfer the energy directly to YFP if the YFP is located in close proximity, thus allowing the emission of higher energy photons by YFP. The measurements of FRET efficiency on a pixel-by-pixel basis will permit monitoring the protein-protein interactions of Eps15 in live cells in real time, and will also permit the precise determination of where in the cell these interactions occur.In addition to providing important specific information about Eps15 interactions with other Eps15 molecules and other proteins, the development of this sensitive assay to study protein:protein interactions in living cells using Eps15 macromolecular complexes as a model system may lead to a breakthrough in the analysis of the interactions involving various other protein complexes and cellular machineries in living cells.