Subsets of Epidermal Growth Factor Receptors during Activation and Endocytosis

Abstract

Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-␥ 1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P) 992 ), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P) 992 receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (␣-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P) 992 receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P) 992 receptors were associated with more SOS, RasGTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated. Receptor protein-tyrosine kinases (RPTKs)1 play an essential role in normal cell growth and neoplasia (1). Following ligand activation, these receptors oligomerize, activate, autophosphorylate, and rapidly endocytose (1-6). Autophosphorylation sites play an important role in signaling as well, since they serve as binding sites for proteins that contain src homology-2 (SH2) domains (7), which in turn propagate the signals of the receptor. SH2 domains are found in many different proteins including enzymes, transcription factors, and adaptor proteins (8, 9). At least eight SH2 proteins bind to the epidermal growth factor (EGF) receptor following activation. Each SH2 domain exhibits specificity based on amino acids surrounding the anchor phosphotyrosine, so the phosphorylation status of the receptor dictates subsequent interactions (10, 11). Interestingly, site-directed mutagenesis on the EGF receptors' autophosphorylation sites has shown that this receptor can be processed differently when these sites are altered. Mutation of Tyr 1068 , Tyr 1148 , or Tyr 1173 slightly prolongs the half-life of this receptor (12, 13), and deletion of all three results in a significant increase in half-life.Several studies have now shown that receptor internalization plays a role in signaling. Upon activation, receptors are endocytosed in clathrin-coated pits, transferred to endosomes, and then multivesicular bodies, which ultimately fuse with lysosomes (5,14,15). Because the autophosphorylation sites and kinase domain of the receptor are...

Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src

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Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

“…In contrast, while a proportion of PI3K-C2␣ was present in phosphotyrosine complexes within minutes, this PI3K isozyme accumulated over a much longer period, about 20 to 40 min, following ligand addition. This difference suggests either differential compartmentalization of the class II PI3K isozymes or a difference in (17,29). EGF stimulation of PI3K activity has been described for a large number of primary cells and cell lines.…”

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells

“…Previous studies demonstrated that EGF treatment promotes the translocation of EGFR from the cell membrane to the perinuclear membrane and nucleus (Emlet et al, 1997;Hsu and Hung, 2007). This nuclear shuttling depends on a novel tripartite type of nuclear localization signal localized in the juxta-membrane domain of EGFR (Hsu and Hung, 2007).…”