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Active EGF receptors have limited access to PtdIns(4,5)P₂ in endosomes: implications for phospholipase C and PI 3-kinase signaling

¹ Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA
² Department of Molecular Pharmacology, Stanford University Medical Center, Stanford, CA 94305-5174, USA

View larger version (61K): [in a new window]   Fig. 1. EGF-receptor-dependent recruitment of SH2 domains to intracellular compartments containing internalized EGF. The tandem SH2 domains of PLC1, fused to enhanced GFP (GFP-SH2), were used as a probe to visualize EGF receptor activation. GFP-SH2-transfected NR6 fibroblasts expressing the wild-type EGF receptor (NR6 WT) were either (A) untreated or (B) stimulated with Texas Red-conjugated EGF (EGF-TR) for 20 minutes. The live cells were subsequently visualized by confocal fluorescence microscopy as described in Materials and Methods. (C) GFP-SH2-transfected, EGF-TR-treated parental NR6 cells, which lack EGF receptors, were used as a control. Scale bar, 20 µm.

View larger version (69K): [in a new window]   Fig. 2. GFP-SH2 is only recruited by autophosphorylation-competent EGF receptors. GFP-SH2-transfected NR6 fibroblasts expressing either the c’1000 EGF receptor truncation mutant (NR6 c’1000; A,B) or the c’1000 EGF receptor with the mutation Y992F (NR6 c’1000F; C,D) were observed in parallel. The cells were either untreated (A,C) or stimulated with EGF-TR for 20 minutes (B,D). Cells were visualized by confocal fluorescence microscopy.

View larger version (58K): [in a new window]   Fig. 3. Intracellular compartments containing internalized EGF lack accessible PtdIns(4,5)P2. The pleckstrin homology domain of PLC1, which is fused to enhanced GFP (GFP-PH), was used as a probe to visualize PtdIns(4,5)P2. GFP-PH-transfected NR6 WT cells were either (A) untreated or (B) stimulated with EGF-TR for 20 minutes. Cells were visualized by confocal fluorescence microscopy. (C) GFP-PH-transfected, EGF-TR-treated parental NR6 cells were used as a control. Scale bar, 10 µm.

View larger version (67K): [in a new window]   Fig. 4. Living NR6 WT cells transfected with GFP-PH were stimulated with EGF. The distribution of GFP was observed in real time using confocal fluorescence microscopy. Images were acquired every 20 seconds, and panels are shown for the indicated times of stimulation.

View larger version (68K): [in a new window]   Fig. 5. The segregation of active EGF receptors from accessible PtdIns(4,5)P2 is not specific to NR6 fibroblasts. A431 cells transfected with GFP-SH2 (A,B) or GFP-PH (C,D) were either untreated (A,C) or stimulated with EGF-TR for 20 minutes (B,D). Cells were visualized by confocal fluorescence microscopy.

View larger version (72K): [in a new window]   Fig. 6. Accessible PtdIns(4,5)P2 is not detected in transferrin-containing trafficking compartments. Texas-Red-conjugated transferrin (Tf-TR) was used as a marker for intracellular compartments of the endocytic pathway. NR6 WT cells transfected with either GFP-SH2 (A,B) or GFP-PH (C,D) were treated for 20 minutes with Tf-TR alone (A,C) or with Tf-TR and unlabeled EGF (B,D). Cells were visualized by confocal fluorescence microscopy.

View larger version (49K): [in a new window]   Fig. 7. PI 3-kinase-dependent phosphorylation of PtdIns(4,5)P2 is not significantly stimulated by internalized EGF receptors. The pleckstrin homology domain of Akt fused to enhanced GFP (GFP-AH) was used to visualize production of PtdIns(3,4,5)P3. GFP-AH-transfected NR6 WT cells were either (A) untreated or (B) stimulated with EGF-TR for 20 minutes. (C) The cell in B was subsequently treated with LY294002 (100 µM, Calbiochem) for 10 minutes and revisualized. (D) GFP-AH-transfected NR6 WT cells were simultaneously treated with Tf-TR and unlabeled EGF for 20 minutes. Cells were visualized by confocal fluorescence microscopy.

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