Altogether, these experiments suggest that Grb2 binding sites participate in coated pit recruitment of EGFRs. Rabbit Polyclonal to Cytochrome c Oxidase 7A2 endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were Narcissoside either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial actions of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane. INTRODUCTION Binding of epidermal growth factor (EGF) to its cell surface receptor (EGFR) initiates an array of signaling events emanating from activation of an intrinsic receptor tyrosine kinase that phosphorylates the receptor itself as well as other intracellular proteins (Schlessinger, 2000 ). These membrane-associated events ultimately lead to altered gene expression Narcissoside in the nucleus. The activation of EGFR by ligand also dramatically changes cellular localization of receptors due to rapid endocytosis of ligand-receptor complexes via clathrin-coated pits. By controlling receptor levels at the cell surface and in endosomes, endocytic trafficking serves as an important determinant of the intensity and duration of EGFR signaling. However, despite extensive studies over last two decades, the molecular mechanisms of clathrin-dependent endocytosis of EGFR are not well comprehended. EGFR mutagenesis revealed that several regions of EGFR molecule can support rapid endocytosis of the receptor. Some of these regions contain well-defined internalization signal motifs (Chang was calculated as linear regression coefficient. Immunoprecipitation and Western Blotting The cells were lysed in Triton X-100/glycerol solubilization buffer as described previously (Jiang and Sorkin, 2002 ). In experiments where ubiquitylation Narcissoside of the EGFR was analyzed, 1% Na deoxycholate and 10 mM values measured under the same conditions (as shown around the left) in NIH 3T3 expressing wild-type or Dc165 mutant EGFR (corresponding to C1021 truncation; Alvarez 0.07/min; Physique ?Physique2A).2A). In contrast, NIH3T3 cells expressing the same mutant displayed high internalization rates. The LL motif (1010/1011) was critical for the low-level internalization of C1022 receptor observed in PAE cells, whereas the Y974RAL and GGQFF1000 motifs had minimal roles in internalization (Physique ?(Figure2B).2B). However, full-length receptors bearing these same mutations alone or in combination were internalized at high rates comparable to wild-type EGFR ( 0.2/min; Physique ?Physique2C).2C). Thus, truncation of last 164 residues appears Narcissoside to uncover cryptic internalization motifs that are not functional in the native receptor in PAE cells. Tyrosines 1068 and 1086 Are Critical for EGFR Internalization The data presented in Physique ?Physique22 suggested that in PAE cells the carboxyl-terminal 164 residues mediate EGFR endocytosis. We, therefore, next focused on defining internalization motifs within the region 1023C1186. Several new truncated EGFR mutants were prepared and constitutively expressed (Physique ?(Figure1A).1A). Analysis of 125I-EGF uptake by these mutants showed that the region between 1063 and 1090 residues is critical for EGFR internalization (Physique ?(Figure3A).3A). Interestingly, the internalization rate Narcissoside of C1063 mutant was even lower than that of C1022, suggesting that residues 1023C1063 may inhibit C1022 internalization by sterically hindering LL or other cryptic motifs. The lower internalization rates of C1063 or comparable mutants compared with C1022 mutant were also observed in other cell types (Chen values were measured in PAE cells stably expressing C1107, C1090, C1072, C1063, or C1022 truncated EGFRs as in Figure ?Physique2.2. (B) values were measured in cells expressing single or double Tyr1068/1086 mutants of full-length or truncated EGFRs (left) as in A. The bars in A and B represent averaged values from multiple experiments with.