Methods to study endocytic trafficking of the EGF receptor

Abstract

Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR.

Keywords: EGF receptor; Endocytosis; Lysosomal degradation; Recycling; Ubiquitin.

FIGURE 1. Time-lapse imaging of UMSCC2 cells stimulated with EGF-Rhodamine (EGF-Rh)

Cells were imaged before and after EGF-Rh (4 ng/mL) stimulation for 15 min. Z-stacks of 20 x–y images (400 nm z-stepsize) of Rhodamine fluorescence were acquired every 1.5 min. 1.5 and 7.5 min images are shown. 3-D volume view image (A) and “3-D view” image (single confocal x–y, y–z, and x–z sections) are presented (B). Scale bars, 10 µm. (See color plate)

FIGURE 2. Effect of Dyngo-4a on EGF-Rhodamine (EGF-Rh) endocytosis in UMSCC2 cells

(A) Cells were incubated 30 min with Dyngo-4a 30 µM or vehicle (DMSO) and then imaged before and after EGF-Rh (4 ng/mL) stimulation as in Figure 1. 3-D volume view images of control and Dyngo-4a pretreated cells that were incubated with EGF-Rh for 5 min are shown. (B) Cells preincubated or not with Dyngo-4a were incubated with EGF-Rh for 15 min in the presence of inhibitor or vehicle, fixed and labeled with wheat germ agglutinin (WGA)-Oregon Green as described in the text. Insets show high magnification images of the peripheral cell regions marked by white rectangles. All image acquisition parameters and intensity scales are the same for corresponding images of control and Dyngo-4a-treated cells. Scale bars, 10 µm. EGF, epidermal growth factor. (See color plate)

FIGURE 3. Internalization of ¹²⁵I-EGF

(A) NIH 3T3/EGFR cells were incubated with 1 ng/mL ¹²⁵I-EGF for 1–6 min at 37 °C, and the amount of surface and internalized radioactivity was determined as described in Section 4.2. (B) UMSCC2 cells were incubated with 30 µM Dyngo-4a in DMEM, and the internalization assay with 1 ng/mL ¹²⁵I-EGF was performed as in (A).

FIGURE 4. Epidermal growth factor receptor (EGFR) ubiquitination

UMSCC2 cells were incubated with or without 4 ng/mL epidermal growth factor (EGF) for 5 min at 37 °C. Cells were solubilized, and EGFR immunoprecipitated as described in Section 4.3.2. EGFR immunoprecipitates and lysates were resolved by electrophoresis, and western blotting was performed using antibodies to ubiquitin, EGFR, and Akt (loading control). The extent of EGFR ubiquitination is calculated as the amount of ubiquitin normalized to the amount of EGFR is shown under the image of the ubiquitin blot.

FIGURE 5. Epidermal growth factor (EGF)-induced EGF receptor (EGFR) degradation

UMSCC2 cells were incubated with 80 ng/mL EGF for 0–24 h. (A) EGFR was detected by western blotting in cell lysates using antibodies 1005. α-actinin is as loading control. (B) Quantification of EGFR immunoreactivity from two experiments including the data presented in (A).

FIGURE 6. Recycling of ¹²⁵I-EGF

(A) Incubation steps in the experimental protocol to measure recycling of ¹²⁵I-EGF. (B) NIH 3T3/EGFR cells were incubated with 5 ng/mL ¹²⁵I-EGF for 5 min at 37 °C, surface ¹²⁵I-EGF was removed by the mild acidic buffer wash, and the recycling assay was performed as described in Section 4.5. The chase incubation medium was supplemented or not with 300 µM primaquine.