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...

The differentiation of chondrocytes and of several other cell types is associated with a switch from the ␣ 6B to the ␣ 6A isoform of the laminin ␣ 6 ␤ 1 integrin receptor. To define whether this event plays a functional role in cell differentiation, we used an in vitro model system that allows chick chondrogenic cells to remain undifferentiated when cultured in monolayer and to differentiate into chondrocytes when grown in suspension culture. We report that: (i) upon over-expression of the human ␣ 6B , adherent chondrogenic cells differentiate to stage I chondrocytes (i.e. increased type II collagen, reduced type I collagen, fibronectin, ␣ 5 ␤ 1 and growth rate, loss of fibroblast morphology); (ii) the expression of type II collagen requires the activation of p38 MAP kinase; (iii) the over-expression of ␣ 6A induces an incomplete differentiation to stage I chondrocytes, whereas no differentiation was observed in ␣ 5 and mock-transfected control cells; (iv) a prevalence of the ␣ 6A subunit is necessary to stabilize the differentiated phenotype when cells are transferred to suspension culture. Altogether, these results indicate a functional role for the ␣ 6B to ␣ 6A switch in chondrocyte differentiation; the former promotes chondrocyte differentiation, and the latter is necessary in stabilizing the differentiated phenotype.Growth factors, cell-extracellular matrix (ECM), 1 and cellcell interactions are the primary determinants of lineage decisions and differentiation events in embryogenesis. These regulatory events involve different types of receptors and may lead to activation of several signaling pathways, such as those mediated by MAP kinases (1-3). Changes in the expression pattern of most of these receptors, including the integrins (heterodimeric ␣␤ receptors involved in cell-ECM and cell-cell interactions), are able to modulate several events associated with cell differentiation (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).The onset of chondrogenesis in developing long bones is characterized by the reduction of intercellular spaces and establishment of extensive cell-cell contacts between mesenchymal chondrogenic cells, i.e. cell condensation (21). Several factors have been shown to play a role in this process, including cell-cell interactions (22-27), composition of the ECM (25, 28 -30), changes in cell shape (31), and response to cytokines (32). Following cell condensation, chondrogenic cells that produce type I collagen, fibronectin (FN), and its integrin receptor ␣ 5 ␤ 1 , differentiate to stage I chondrocytes that express type II collagen and eventually to stage II hypertrophic chondrocytes, characterized by type II and X collagen production (12).Most of these events can be reproduced in vitro in a tissue culture model system that allows condensation and differentiation of chick embryo tibiae chondrogenic cells (12,(33)(34)(35). These cells, which adhere to tissue culture dishes and display a fibroblast-like phenotype (pre-chondrogenic cells), proliferate and secrete type I collagen...