Related Experiment Videos
Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts
J M Haugh1, A C Huang, H S Wiley
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Abstract:
Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc.Grb2.Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-alpha, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.
Insights
EGFR endocytosis regulates Ras activation. Both cell surface and internal EGFRs equally activate Shc, but receptor trafficking limits Ras-GTP generation, a process reversible by blocking MEK feedback.
Area of Science:
- Cell biology
- Molecular signaling
- Receptor tyrosine kinases
Background:
- Epidermal growth factor receptor (EGFR) activation initiates signaling cascades controlling cell growth and motility.
- Ras GTPase activation is crucial for these downstream cellular responses.
- Receptor down-regulation and feedback loops involving MEK regulate Ras activity.
Purpose of the Study:
- To investigate the role of EGFR endocytosis in regulating Ras activation.
- To determine if activated receptors within endosomes can stimulate Ras guanine nucleotide exchange.
- To understand the compartmentalization of Ras signaling.
Main Methods:
- Utilized differences in postendocytic signaling of EGF and TGF-α ligands.
- Examined Shc recruitment and phosphorylation in NR6 fibroblasts expressing wild-type EGFR.
- Assessed Ras-specific guanine nucleotide exchange and Ras-GTP levels.
- Employed the MEK inhibitor PD098059 to block feedback desensitization.
Main Results:
- Activated EGFR at the cell surface and in internal compartments equally contributed to Shc recruitment and phosphorylation.
- Receptor trafficking led to a depression of Ras-specific guanine nucleotide exchange and Ras-GTP levels.
- Blocking MEK feedback with PD098059 maintained Ras activation, irrespective of EGFR subcellular location.
Conclusions:
- EGFR endocytosis plays a significant role in regulating Ras activation.
- Activated EGFR in endosomes can participate in Shc-mediated signaling.
- Receptor trafficking and MEK-dependent feedback differentially regulate specific downstream signaling pathways.