Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrincoated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization. INTRODUCTIONOn engagement of receptor tyrosine kinases (RTKs) by their cognate ligands, their intrinsic kinase activity is stimulated with ensuing receptor autophosphorylation and recruitment/activation of the signal transduction machinery, in turn responsible for several effector functions. Concurrently, activated receptors trigger their own endocytosis, whose ultimate goal is to extinguish signaling through removal of receptors from the cell surface (Carpenter, 2000). In the case of the epidermal growth factor (EGF) receptor (EGFR) the localization of the two processes is well characterized (Jorissen et al., 2003). Signaling occurs within specialized membrane microdomains, lipid rafts (Simons and Toomre, 2000;Maxfield, 2002), whereas endocytosis occurs mostly through the clathrin-coated pits (CCPs; Conner and Schmid, 2003).Membrane rafts are cholesterol-and sphingolipid-rich membrane regions characterized by higher order and lower buoyant density than bulk plasma membrane (Simons and Toomre, 2000;Sprong et al., 2001;Kusumi et al., 2004). These structures are also characterized by their insolubility in some detergents at 4°C (DRM, detergent resistant membranes; Brown and Rose, 1992). Several transmembrane receptors have been reported to associate with membrane rafts (Cheng et al., 1999;Krauss and Altevogt, 1999;Mineo et al., 1999;Lamaze et al., 2001;Giurisato et al., 2003), including the EGFR . The association of receptors with lipid rafts is thought to be functional to the activation of signaling cascades (Cheng et al., 1999;Waugh et al., 1999;Drevot et al., 2002;Matveev and Smart, 2002;Pierce, 2002;Stoddart et al., 2002;del Pozo et al., 2004). Accordingly, specific signaling (Gingras et al., 1998;Iwabuchi et al., 1998;Michaely et al., 1999;Kindzelskii et al., 2004) and adaptor proteins (e.g., shc and grb2; Biedi et al., 2003;Ridyard and Robbins, 2003;Yang et al., 2004) have been found associated to rafts. However, lipid rafts are rather small, possibly containing only few molecules (P...

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Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.