Gab1 signaling is regulated by EGF receptor sorting in early endosomes.

Although combinatorial signaling through the ErbB network is implicated in certain types of human cancer, the specifics of how particular receptors contribute to the transformed phenotype are not well understood. The goal of this study was to identify epidermal growth factor (EGF) receptor-dependent cell signaling abnormalities specifically associated with mutations in a previously described 679-LL lysosomal sorting signal, which restrict ligand-dependent receptor downregulation by promoting recycling. Importantly, the 679-LL signal is not conserved in any of the other members of the ErbB receptor family suggesting its physiological function may be tightly regulated during EGF receptor-dependent signaling. Our data indicate that cells expressing receptors with an inactive 679-AA signal are rapidly transported to Rab4+ early endosomes after they are internalized in contrast to wild-type receptors that are localized to early endocytic antigen 1 (EEA1)+ early endosomes. Divergent trafficking in early endosomes is associated with prolonged activation of p44/42 mitogen-activated protein kinases (MAPK) but not Akt. Gab1 appears to be the critical signaling molecule facilitating prolonged MAPK signaling, and activated Gab1 is recruited to early endosomes in 679-AA receptor-expressing cells. Activated Gab1 is also recruited to early endosomes in breast cancer cells characterized by high levels of EGF receptor-ErbB2 heterodimers, suggesting 679-AA expressing cells recapitulate certain aspects of EGF receptor signaling and transformation by activated ErbB2. Phosphatidylinositol 3-kinase (PI3K)-dependent membrane translocation known to be important for maintaining Gab1 activity in other settings was dispensable. We conclude that 679-LL has dual functions in EGF receptor trafficking and threshold signaling through a subset of signaling molecules including p44/42 MAPK and Gab1.

E3-13.7 integral membrane proteins encoded by human adenoviruses alter epidermal growth factor receptor trafficking by interacting directly with receptors in early endosomes.

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7-mediated down-regulation in early endosomes. Receptor-viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675-697 are required for E3-13.7-mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments.

Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.

A leucine-based determinant in the epidermal growth factor receptor juxtamembrane domain is required for the efficient transport of ligand-receptor complexes to lysosomes.

Ligand binding causes the epidermal growth factor (EGF) receptor to undergo accelerated internalization with eventual degradation in lysosomes. The goal of this study was to investigate the molecular basis of endocytic sorting, focussing on post-internalization events. We have identified a sequence located between amino acid residues 675 and 697, encompassing a dileucine motif at residues 679 and 680, that enhances endosome-to-lysosome transport when conformational restraints in the EGF receptor carboxyl terminus are removed by truncation. The same dileucine motif is also necessary for efficient lysosomal transport of ligand-occupied full-length EGF receptors. A L679A,L680A substitution diminished the degradation of occupied full-length EGF receptors without affecting internalization but had a significant effect on recycling. Rapid recycling of mutant receptors resulted in reduced intracellular retention of occupied EGF receptors and delayed down-regulation of cell surface receptors. We propose that the L679A,L680A substitution acts primarily to impair transport of ligand-receptor complexes through an early endosomal compartment, diverting occupied receptors to a recycling compartment at the expense of incorporation into lysosome transport vesicles. We also found that mutant receptors with truncations at the distal half of tyrosine kinase domain (residues 809-957) were not efficiently delivered to the cell surface but were destroyed in an endoplasmic reticulum-associated degradative pathway.

The cytoplasmic juxtamembrane domain of the epidermal growth factor receptor contains a novel autonomous basolateral sorting determinant.

The epidermal growth factor receptor (EGFR) is localized at the basolateral membrane of most epithelial cells in vivo and in cell lines used to study membrane protein sorting. The goal of this study was to define the molecular basis of polar EGFR membrane expression using the Madin-Darby canine kidney cell model. We have identified a 23-amino acid segment located near the cytoplasmic face of the membrane spanning domain (residues Lys-652 to Ala-674) that is necessary and sufficient for targeting EGFRs from the trans-Golgi network directly to the basolateral plasma membrane. Furthermore, the sequence between residues Lys-652 and Ala-674 is sufficient to direct the extracellular domain of an apical membrane protein, decay accelerating factor, to the basolateral membrane. In the absence of this cytoplasmic basolateral sorting signal, information within the extracellular ligand binding domain is sufficient to target EGFRs from the trans-Golgi network directly to the apical plasma membrane. The EGFR basolateral sorting determinant does not have sequence and structural requirements common to most basolateral membrane proteins and does not overlap any of the known EGFR endocytic signals. This 23-residue sequence lies in a predicted amphipathic helical structure, leading us to postulate that hydrophobic and/or electrostatic interactions may be important for activity of this autonomous basolateral sorting determinant.

Involvement of opioid peptides from casein on reticular motility and digesta passage in steers.

Experiment 1 determined the effects of incremental abomasal casein infusion on reticular motility and digesta passage of mature steers in a 4 x 4 Latin square study. Casein solutions were infused at 0, 1.25, 2.5, and 5.0% (wt/vol) for 3 h at 16.7 ml/min. Steers were fed for maintenance every 2 h. Casein resulted in a linear decrease in frequency, duration, and amplitude of reticular contractions measured manometrically. Total area of reticular contractions from physiographic tracings decreased 10.4 to 18.5%. Ruminal DM contents decreased 4.5 to 7.5%. Experiment 2 characterized the effects of casein on reticular contractions to be mediated by opioid peptides. Steers were infused abomasally (4 h) with either a 5% (wt/vol) solution of casein or its hydrolysate with or without a predose of naltrexone as an opiate antagonist (.5 mg/kg of BW) in a 2 x 2 factorial. Both casein and hydrolysate reduced frequency and duration of reticular contractions. Hydrolysate decreased contraction frequency within 30 min of infusion and 60 min prior to effects of casein. Naltrexone reversed the effects of casein and accentuated the effects of hydrolysate. Ruminal liquid outflow was decreased 19.7% with hydrolysate. Postruminal infusion of casein or its hydrolysate resulted in casomorphin-like activity on reticular motility and digesta passage.