Effect of Epidermal Growth Factor Receptor Internalization on Regulation of the Phospholipase C-γ1 Signaling Pathway

Abstract

The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-␣ (TGF␣) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGF␣-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-␥1 (PLC-␥1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGF␣ stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) hydrolysis over those stimulated by TGF␣. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP 2 hydrolysis, PLC-␥1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-␥1 phosphorylation and PIP 2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-␥1 to its substrate in endocytic trafficking organelles.Cell signaling events mediated by epidermal growth factor receptor (EGFR) 1 regulates survival, proliferation, migration, and differentiation of many cell types. At least five ligands are known to activate EGFR, including epidermal growth factor (EGF) and transforming growth factor ␣ (TGF␣). Progress has been made in the last two decades in elucidating structurefunction relationships for EGFR and other receptor tyrosine kinases, particularly in how signal transduction is modulated by self-phosphorylation of cytoplasmic tyrosine residues (1). This permits access to the kinase domain of EGFR (2) and allows the receptor to bind signaling proteins containing modular Src homology 2 (SH2) and phosphotyrosine-binding domains (3, 4). Such interactions can affect the activity of the bound protein through transmission of conformational changes, enhancement of tyrosine phosphorylation, and/or localization in proximity to membrane-associated target molecules. One of the prominent signaling proteins activated by EGFR is the ␥1 isoform of phospholipase C (PLC) (5). This enzyme, which has two SH2 domains, catalyzes the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ), generating the second messengers diacylglycerol and inositol triphosphate and liberating PIP 2 -bound proteins (6). PLC-␥1 activity is positively modulated ...

Association of Villin with Phosphatidylinositol 4,5-Bisphosphate Regulates the Actin Cytoskeleton

“…Several recent studies (60,61) have demonstrated that changes in phosphoinositide levels, specifically PIP 2 , and concomitant changes in the actin dynamics are essential to some cell surface events including motility. In fact, peptides encompassing the PIP 2 binding domain of an actin-binding protein of the villin superfamily (gelsolin) have been shown to increase cell motility in fibroblasts (62).…”

Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 in Fibroblasts

“…Surface Titration Protocol-This stimulation procedure allows the numbers of activated EGFR at the plasma membrane and in intracellular compartments following endocytosis to be varied independently, as described previously (32) and illustrated in Fig. 1.…”

“…Lysates were clarified by centrifugation, diluted to various extents in blocking buffer supplemented with 1 mM sodium orthovanadate, and incubated in the antibody-coated wells for 1 h at 37°C. The amount of associated phosphotyrosine was determined using alkaline phosphatase-conjugated RC20 anti-phosphotyrosine antibody (Transduction Laboratories) and p-nitrophenyl phosphate (Sigma) substrate as described previously (32).…”

“…On the other hand, trafficking of the EGFR between the surface and intracellular compartments requires about 20 min to reach a quasi-steady state in NR6 WT cells (32,33). Although receptor internalization may play a role in the deactivation of Ras, it is also known that a MEK-dependent feedback loop, acting downstream of receptor activation and upstream of Ras activation, is a potent regulatory mechanism in this pathway (23)(24)(25)(26)(27).…”

“…However, the fact that Ras is a membrane-associated protein suggests that it might be compartmentalized. For example, we previously showed that activated EGFR in internal compartments effectively participate in the tyrosine phosphorylation of PLC-␥1, but not in the hydrolysis of its membrane lipid substrate phosphatidylinositol (4,5)-bisphosphate (32). We therefore endeavored to determine whether active, internalized EGFR could participate in the activation of Ras.…”