Plasma membrane receptors can be endocytosed through clathrindependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR͞ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.internalization ͉ rafts ͉ caveolae ͉ ubiquitination ͉ ubiquitin-interacting motif U biquitination is a posttranslational modification whereby substrate proteins are conjugated to a short highly conserved peptide, ubiquitin (Ub), through the action of Ub ligases (E3 enzymes). Polyubiquitination, in which a chain of Ub is appended, targets proteins to proteasomal degradation (1). However, when a single Ub moiety is appended (monoubiquitination), the modification functions as a signaling device through interactions with intracellular proteins harboring Ub-binding domains, such as the Ub-interacting motif (UIM) (2). In yeast, monoubiquitination has been known to act as an internalization signal for quite some time (3). In mammals, however, this connection has remained more elusive.We are interested in the mechanisms of internalization of receptor tyrosine kinases and, in particular, the epidermal growth factor receptor (EGFR). The EGFR is monoubiquitinated at multiple sites (4) through the action of the E3 enzyme Cbl. Although there is consensus on the function of Cbl and receptor ubiquitination in intracellular sorting of the EGFR, their role in the internalization step of endocytosis is less clear (5, 6). To gain insight into this issue, we generated a chimera in which the extracellular and transmembrane domains of the EGFR are fused to a mutant Ub (Ubmut), unable to form polyUb chains (EGFR͞Ubmut). With this chimera, we showed that ubiquitination is sufficient for internalization (4). The present studies were undertaken to elucidate the molecular mechanisms through which receptor ubiquitination directs internalization. Materials and MethodsTransfection and Biochemical Studies. Transfections were performed by using Lipofectamine or Oligofectamine (Invitrogen). For biochemical experiments, cells were serum-starved and then stimulated with EGF (100 ng͞ml, unless otherwise indicated) at 37°C. Lysis, immunoprecipitation, and immunoblot...

Pathways that govern stem cell (SC) function are often subverted in cancer. Here, we report the isolation to near purity of human normal mammary SCs (hNMSCs), from cultured mammospheres, on the basis of their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we prospectively isolated SCs from the normal gland and from breast tumors. Poorly differentiated (G3) cancers displayed higher content of prospectively isolated cancer SCs (CSCs) than did well-differentiated (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in CSCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their CSC content.