Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

Abstract

Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca²⁺, or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca²⁺- and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca²⁺ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes.

FIGURE 1:

PLCγ1 is required for EGF but not Tfn internalization. RPE cells were treated with siRNA targeting PLCγ1 or nontargeting siRNA, followed by measurement of EGF (A), Tfn (B), or Tfn internalization in EGF-stimulated cells (C). Shown are the means ± SE for n > 3 independent experiments; *, p < 0.05.

FIGURE 2:

EGF stimulation elicits a PLCγ1-dependent increase in cytosolic Ca²⁺. (A) Biochemical PIP2 measurement (means ± SE) in cells treated with EGF (5 or 20 ng/ml, 5 min) or ionomycin (10 μM, 20 min) as indicated. n = 3; *, p < 0.05. (B) RPE cells transfected with cDNA encoding the wild-type PH domain of PLCδ1 fused to mCherry (mCh-PH WT) or a similar fusion with a K32A/W36A mutant PH domain (unable to bind PIP2; Antonescu et al., 2011). Shown are representative micrographs obtained by wide-field epifluorescence or TIRF microscopy, as indicated and the means ± SE of the TIRF/epifluorescence ratio of the mCherry signal, indicative of membrane binding by the PH probe. (C–G) RPE cells were treated with siRNA targeting PLCγ1 or nontargeting siRNA, or treated with 3 μM XeC for 20 min or left untreated (control). RPE cells were then treated with Fluo4-AM for 30 min. Shown in C and E are representative images obtained by wide-field epifluorescence of Fluo4 obtained before further treatments (basal), after stimulation with 5 ng/ml EGF for 5 min, and then again after subsequent treatment with 10 μM ionomycin for 5 min, as indicated. Scale bars: 20 μm. Also shown are the EGF-stimulated (D, F) or ionomycin triggered (G) gains (means ± SE) in Fluo4-AM fluorescence within cells in the various conditions examined. n > 3, *, p < 0.05.

FIGURE 3:

IP3 receptor and cytosolic Ca²⁺ are required for EGF but not Tfn internalization. RPE cells were treated with 3 μM XeC for 30 min, 10 μM BAPTA-AM for 15min, or left untreated (control) as indicated, followed by measurement of EGF (A, D), Tfn (B, E) or Tfn internalization in EGF-stimulated cells (C, F). Shown are the means ± SE for n > 3 independent experiments; *, p < 0.05.

FIGURE 4:

PKC, but not calcineurin, is selectively required for EGF internalization. RPE cells were treated with 10 μM CsA for 30 min or 1 μM BIM for 30 min, or left untreated (control), followed by measurement of EGF (A, B), Tfn (C), or Tfn internalization in EGF-stimulated cells (D). Shown are the means ± SE for n > 3 independent experiments; *, p < 0.05.

FIGURE 5:

EGF and Tfn are recruited to largely distinct CLSs. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa) were treated with 20 ng/ml rhodamine-EGF (rho-EGF) and 10 μg/ml A647-Tfn for 5 min or the indicated time, followed by immediate fixation. (A) Shown are representative micrographs obtained by TIRF-M. Scale bars: 10 μm (top row); 5 μm (bottom row, corresponding to enlarged images of the region shown in the merged image of the top row). White circles depict CLSs that are positive for EGF but devoid of Tfn, and purple circles depict CLSs that are positive for Tfn but not EGF. (B, C) CLSs were subjected to automated detection and analysis as described in Materials and Methods. Shown in B is a two-dimensional histogram of the normalized EGF and Tfn fluorescence intensities in each CLS cohort. Shown in C are median (bar) and 25th/75th percentiles (boxes) of the proportions of CLSs that are positive for EGF (but not Tfn), Tfn (but not EGF), or both EGF and Tfn. The number of CLSs and cells analyzed, respectively, for each condition are as follows: 10 ng/ml rhodamine-EGF and 10 μg/ml A647-Tfn: 70,124 and 57; and 10 ng/ml rhodamine-EGF and 10 μg/ml A647-Tfn: 46,240 and 37; from a minimum of three independent experiments in each condition. (D–F) Images obtained by spinning-disk confocal microscopy, corresponding to ventral, middle, and top z-sections of cells (see Supplemental Figure 5 for representative images) were subjected to automated detection and analysis as described in Materials and Methods. Shown are median (bar) and 25th/75th percentiles (boxes) of the proportions of CLSs in the ventral (D), middle (E), and top (F) z-sections that are positive for EGF (but not Tfn), Tfn (but not EGF), or both EGF and Tfn. The number of CLSs analyzed and cells for each condition (from three independent experiments) are provided in the legend for Supplemental Figure 5.

FIGURE 6:

Cytosolic Ca²⁺ and PKC regulate CLSs containing EGFR but not those harboring TfR. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa), treated with various inhibitors as in Figures 3 and 4: 3 μM XeC for 30 min, 10 μM BAPTA-AM (BA) for 15 min, 1 μM BIM for 30 min, or left untreated (control), and then treated with A555-EGF (A) or A555-Tfn (B) for 5 min. Shown are representative micrographs obtained by TIRF-M. Scale bar: 5 μm. Full image panels are shown in Supplemental Figure 7. (C–F) TIRF-M images were subjected to automated detection of CLSs, followed by quantification of mean A555-conjugated ligand fluorescence intensity therein (C, E). CLSs were sorted into A555-EGF-enriched or A555-Tfn-enriched cohorts, followed by quantification of the mean eGFP-CLC within each CLS cohort (D, F). For C–F, the overall median of the cellular means (bar) and 25th/75th percentiles (boxes) are shown. The number of CLSs and cells analyzed, respectively, for each condition are as follows: EGF control: 14,114 and 114; EGF XeC: 11,322 and 98; EGF BAPTA-AM: 15,786 and 104; EGF BIM: 3860 and 46, Tfn control: 16,983 and 117; Tfn XeC: 10,838 and 98; Tfn BAPTA-AM: 8868 and 64; Tfn BIM: 3718 and 44; from a minimum of three independent experiments in each condition.

FIGURE 7:

Cytosolic Ca²⁺ selectively controls initiation and assembly of CCPs harboring EGFR. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa) were pretreated with 10 μM BAPTA-AM for 15 min, and then treated with either 20 ng/ml rhodamine-EGF (Rho-EGF) or treated with A647-Tfn during time-lapse imaging by TIRF-M. (A, B) Single-frame representa­tive fluorescence micrographs. Scale bar: 5 μm. Time-lapse TIRF-M image series of cells treated with Rho-EGF (C–E) or A647-Tfn (F–H) were subjected to automated detection, tracking, and analysis of CLSs as described in Materials and Methods, allowing identification of sCLSs and bona fide CCPs as EGF+ or Tfn+, as appropriate. (C–D, F–G) Median, 25th/75th percentiles (boxes) and Tukey range (whiskers) for the initiation rate of ligand-positive sCLSs (C, F) or ligand-positive CCPs (D, G) are shown. (E, H) Mean eGFP-CLCa fluorescence intensity grouped into CCP lifetime cohorts; error bars reflect cell-to-cell variation. The number of total CLS trajectories, CCP trajectories, and cells for each condition are (respectively): rho-EGF treated (control, DMSO): 24,737, 16,621, and 18; rho-EGF treated (BAPTA-AM treated): 33,604, 14,731, and 24; A647-Tfn (control, DMSO) 32,693, 22,008, and 24; A647-Tfn (BAPTA-AM) 35,287, 20,862, and 25. The breakdown of ligand-positive sCLSs and CCPs is as follows: 5.0 ± 0.7% (control) and 8.5 ± 0.5% (BAPTA-treated) of sCLSs are EGF+; 13.2 ± 0.9% (control) and 10.9 ± 0.9% (BAPTA-treated) of CCPs are EGF+; 3.1 ± 0.4% (control) and 4.0 ± 0.7% (BAPTA-treated) of sCLSs are Tfn+; 13.6 ± 1.9% (control) and 13.2 ± 1.9% (BAPTA-AM) of CCPs are Tfn+.

FIGURE 8:

mCherry-Sjn1 is depleted from CLSs upon EGF stimulation in a Ca²⁺- and PKC-dependent manner. RPE cells stably expressing GFP-CLCa were transfected with mCherry-tagged Sjn1 (mCh-Sjn1, 170-kDa isoform), and then treated with 10 μM BAPTA-AM for 15 min, 1 μM BIM for 30 min, or left untreated (control), followed in some samples by stimulation with 5 ng/ml EGF, as indicated, then by immediate fixation. (A) Shown are representative micrographs obtained by TIRF-M. Scale bars: 10 μm. Full-image panels, including corresponding epifluorescence images, are shown in Supplemental Figure 10. (B) TIRF-M images were subjected to automated detection of CLSs, followed by quantification of the mean mCh-Sjn1 fluorescence intensity therein (normalized to total mCh-Sjn1 expression determined from epifluorescence images). Shown in B are median (bar) and 25th/75th percentiles (boxes) of mCh-Sjn1 fluorescence intensity within CLSs in each condition. The number of CLSs and cells analyzed, respectively, for each condition are as follows: basal, control (no drug): 43,047 and 50; basal, BAPTA-AM: 38,919 and 44; basal, BIM: 35,556 and 46; EGF, control (no drug): 55,482 and 55; EGF, BAPTA-AM: 28,644 and 43; EGF, BIM: 29,637 and 38; from three independent experiments.

FIGURE 9:

PLCγ1 is required for clathrin-dependent EGFR signaling. RPE cells or RPE cells stably expressing HER2 (RPE-HER2 cells) were subjected to siRNA silencing of PLCγ1and treated with 5 ng/ml EGF for 5 min as indicated, followed by Western blotting of whole-cell lysates with antibodies to detect phosphorylated proteins. Shown are representative immunoblots and the mean ± SE signal intensity for (A) pAkt and pGab1 in RPE cells (not expressing HER2), (B) pEGFR and pErk in RPE cells (not expressing HER2), and (C) pAkt in RPE-HER2 cells. *, p < 0.05.