Plasma membrane transporters belonging to the family of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters play an important role in terminating the activity of the monoamine neurotransmitters and of ␥-aminobutyric acid (see Ref. 1). Thus, reuptake of dopamine (DA) 1 from the synaptic cleft by the dopamine transporter (DAT) serves as the major mechanism for terminating dopaminergic neurotransmission in the brain (2). Because the efficiency of DA removal depends on the number of DAT molecules expressed at the plasma membrane, trafficking processes that control transporter distribution in the cell represent a potentially important mechanism by which neurotransmission could be regulated. Newly synthesized DAT acquires glycosylation in the endoplasmic reticulum (ER) and Golgi complex and is then trafficked to the plasma membrane. Typically, a large pool of mature DAT molecules are found at the cell surface of dopaminergic neurons and when DAT is heterologously expressed in tissue culture cells. However, DAT localization can be altered rapidly. Acute exposure of cells to either phorbol esters or substrates reduces the number of plasma membrane DATs and thus DAT function, and this reduction is due to acceleration of DAT endocytosis through a dynamin-dependent mechanism (3-6). Recently, it has been shown that DAT can interact with the anchoring protein PICK 1 and the adaptor protein 8). However, in general, the molecular mechanisms controlling DAT trafficking are not yet well understood.The DAT molecule is predicted to have 12 membrane-spanning sequences with both amino and carboxyl termini oriented intracellularly. The specific function of DAT transmembrane (TM) motifs and termini are not well established. TM domains may play a role in intra-and intermolecular interactions. Many membrane receptors and other integral membrane proteins require dimerization or higher oligomerization for their activity. Several lines of evidence have suggested that monoamine transporters are also dimers or oligomers. Results with dominant negative forms of the serotonin transporter (SERT) and norepinephrine transporter were consistent with this idea (9, 10). Oligomerization of SERT was demonstrated directly by co-immunoprecipitation (11). The results of fluorescence resonance energy transfer (FRET) studies further confirmed that SERT and a ␥-aminobutyric acid transporter (GAT-1) are homo-oligomers (12, 13). Early radiation inactivation studies also suggested that DAT exists as a dimer or oligomer (14,15). Recently, the potential for DAT to exist as dimer or higherorder oligomer in the plasma membrane has been demonstrated using chemical cross-linking (16). More recently, Torres and co-workers (17) reported detection of DAT oligomerization by co-immunoprecipitation. In general, the mechanisms and functional roles of oligomerization of monoamine transporters remain to be defined. However, it has been proposed that SERT oligomerization may be important for its transport activity (9). Oligomerization may be also required for proper traffic...

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer . This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.Many cellular processes are governed by multi-component molecular machineries that rely on dynamic and highly coordinated protein-protein interactions. For example, assembly of protein complexes during signal transduction processes increases the speed of enzymatic reactions, ensures the specificity of signaling and targets signaling molecules to proper intracellular compartments. During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments 4-7 . FRET-based genetically encoded biosensors for second messengers, protein phosphorylation and activity of small GTPases have provided insights into the spatial and temporal regulation of signaling processes [8][9][10][11][12] .The combination of enhanced cyan (CFP) and yellow (YFP) fluorescent proteins has proven to be most effective in many FRET studies. Cloning of Anthozoa fluorescent proteins, such as red DsRed 13,14 and far-red HcRed 15 , has expanded the in vivo applications of FRET, but a major limitation of Anthozoa proteins for FRET applications is their obligate oligomerization 16 . Recent generation of a monomerized mutant of DsRed, monomeric red fluorescent protein 1 (mRFP) 17 , provides the opportunity to generate functional monomeric fusion proteins and to use mRFP as a FRET acceptor with proteins fused to GFP or its mutants.Various methods of FRET measurements have been used to visualize protein-protein interactions 18 . The general limitation of these methods is that they only permit the analysis of interactions between two proteins. To analyze multi-component signaling complexes, a method to measure interactions betwe...

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Ligand-induced receptor-mediated endocytosis plays a central role in regulating signaling conveyed by tyrosine kinase receptors. This process depends on the recruitment of the adaptor protein 2 (AP-2) complex, clathrin, dynamin, and other accessory proteins to the ligand-bound receptor. We show here that besides AP-2 and clathrin, two other proteins participate in the endocytic process of the insulin-like growth factor receptor (IGF-1R); they are EHD1, an Eps15 homology (EH) domain-containing protein 1, and SNAP29, a synaptosomal-associated protein. EHD1 and SNAP29 form complexes with ␣-adaptin of AP-2 and co-localize in endocytic vesicles, indicating a role for them in endocytosis. EHD1 and SNAP29 interact directly with each other and are present in complexes with IGF-1R. After IGF-1 induction, EHD1 and IGF-1R co-localize intracellularly. Overexpression of EHD1 in Chinese hamster ovary cells represses IGF-1-mediated signaling, as measured by mitogen-activated protein kinase phosphorylation and Akt phosphorylation, indicating that EHD1 plays a role as a down-regulator in IGF-1 signaling pathway.Tyrosine kinase receptors convey signals that affect central cellular processes of proliferation, differentiation, metabolism, and apoptosis (1). Upon binding to their cognate ligand, the receptors undergo autophosphorylation within specific motifs that result in extensive recruitment of accessory proteins (2-4). This process promotes rapid signaling after sequestration of the activated receptor via ligand-induced receptor-mediated endocytosis (5). After internalization, tyrosine kinase receptorcontaining vesicles are directed through vesicular structures into endosomal compartments that are further sorted on microtubular tracks to lysosomes or proteasomes for degradation of the receptors (6 -8). Clathrin-mediated endocytosis is a well studied process whereby essential components of the basal endocytic machinery like the AP-2 1 adapter complex, clathrin, endophilin, and dynamin are recruited to the plasma membrane to promote the formation of clathrin-coated vesicle (9 -12). The dynamic assembly of these complexes is mediated through various protein recognition modules. One of the modules is the EH domain that was first identified as a 100-amino acid sequence repeated 3 times in the N terminus of the EGF receptor pathway substrate Eps15 (13-18). The central region of Eps15 shares the characteristic heptad repeats of coiled-coil proteins, and the C terminus contains a prolin-rich region and a repeated DPF motif (16,17). Intracellular localization to plasma membrane pits and vesicles, interaction with prominent proteins of the basal endocytic machinery like ␣-adaptin of AP-2, and interactions through the EH domain with other proteins harboring the NPFXD sequence like epsin have implicated Eps15 in receptor-mediated endocytosis (18 -22). Moreover, functional inhibition of Eps15 by antibodies or dominant negative mutants abrogated endocytosis of EGF and transferrin, therefore supporting the notion that Eps15 mediates endocytos...