Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

Insecure attachment relations have been theorized to play a significant role in the development of depressogenic modes of adaptation and to thus form a vulnerability factor for the emergence of depressive disorder in children. This study examined security of parent and peer attachment among four groups of early adolescents: clinically depressed, nondepressed psychiatric controls, nonpsychiatric controls, and adolescents with resolved depression. Depressed adolescents reported significantly less secure parent attachment than either of the control groups, and less secure peer attachment than the nonpsychiatric control group. Attachment security of adolescents with resolved depression was on a par with the nonpsychiatric control group. Among all psychiatric patients, security of attachment to parents was negatively correlated with severity of depression according to interview and self-report ratings. Less secure attachment to parents, but generally not to peers, was also related to more maladaptive attributional styles, presence of separation anxiety disorder, and history of suicidal ideation.

Activated receptor tyrosine kinase (RTK) receptors are rapidly internalized and eventually delivered to the lysosomes. Although ligand-induced endocytosis was originally thought to be a mechanism of receptor inactivation, many studies suggest that receptors remain active within endosomes. This review discusses the role that internalized signaling complexes may play in different RTK systems including recent data on how ubiquitination may regulate this process. In general, it appears that some receptor systems have evolved to enhance endosomal signaling, as is the case for TrkA and NGF. In contrast, the insulin receptor system appears to limit the extent of endosomal signaling. The EGFR system is the intermediate example. In this case, some signals are specifically generated from the cell surface while others appear to be generated from within endosomes. This may act as a mechanism to produce ligand-specific signals. Thus, trafficking could play diverse roles in receptor signaling, depending on the specific cell and tissue type. Ever since the discovery that hormone and growth factor receptors are internalized following ligand binding, scientists have asked 'why?' Historically, receptors were thought to reside exclusively at the cell surface where they were in a position to sample the extracellular environment. The notion of an activated receptor being rapidly transferred to an intracellular compartment was intriguing and seemed full of interesting possibilities with respect to receptor function. The ensuing decades have witnessed many efforts to unravel the role of endocytosis in receptor signaling, but many issues remain unresolved. Certainly, early hypotheses that endocytosis delivered activated receptors to the nucleus have found little support. However, our deepening understanding of the complexities of signaling networks has provided many new and interesting hypotheses. In addition, our improved understanding of endocytosis in general has provided a valuable context for understanding regulated receptor trafficking. In this review, we will cover some of the most recent findings regarding the trafficking of receptor tyrosine kinases (RTKs) and the role this plays in regulating signal transduction.

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

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Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti–EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti–EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.

This study explores whether cognitive attributes differentiate depressed children from those with other psychiatric disorders. The subjects were 108 children from 7 to 17 years of age. Forty-seven children were diagnosed as currently depressed, 30 as having had an episode of major depression within the last year (depressed-resolved), and 31 with diagnoses other than depression (nondepressed). The subjects completed the Piers-Harris Children's Self-Concept Scale, the Children's Hopelessness Scale, the Nowicki-Strickland Children's Locus of Control Scale, the Children's Attributional Styles Questionnaire, and the Children's Depression Inventory. The depressed children endorsed significantly lower self-esteem, more hopelessness, a more externalized locus of control, and a more depressive attributional style than the depressed-resolved or the nondepressed children. Thus, a depressive cognitive style can be documented in clinically depressed young people.Depression in children and adolescents has received considerable attention over the past 10 years. Most of these research efforts have focused on diagnostic questions (Cantwell & Carl-