Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation

Abstract

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.

FIG. 1.

The EGFR is organized into nanoclusters on the plasma membrane. (A) Plasma membrane sheets generated from BHK cells expressing EGFR-GFP were labeled with anti-GFP antibody conjugated to 5-nm gold particles. The spatial distribution of the gold labeling was analyzed using Ripley's K function. Maximum L(r) − r values above the 99% CI for complete spatial randomness indicate clustering at the value of r (supr). Univariate K functions are weighted means (n = 15) standardized on the 99% CI. (B) BHK cells expressing EGFR-GFP were left untreated or treated with either 1% methyl-β-cyclodextrin for 60 min or 1 μM latrunculin A for 5 min. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 6) standardized on the 99% CI. Significant differences from the control EGFR pattern were assessed using bootstrap tests. Treatment with latrunculin A did not significantly alter EGFR nanocluster formation (P = 1). However, treatment with methyl-β-cyclodextrin significantly decreased EGFR nanoclustering (P = 0.001).

FIG. 2.

EGFR nanocluster formation is induced by EGF stimulation. BHK cells expressing EGFR-GFP were serum starved and then stimulated with 50 ng/ml EGF for the indicated times. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 10) standardized on the 99% CI. Significant differences from the control unstimulated EGFR pattern were assessed using bootstrap tests. (A) Stimulation with EGF resulted in a significant increase in EGFR nanocluster formation at all time points (P = 0.001). (B) Stimulation with EGF for 10 and 20 min led to a significant increase in EGFR nanoclustering compared to that obtained with the unstimulated control (P = 0.003 and P = 0.001, respectively). However, EGFR nanoclustering was not significantly different from unstimulated EGFR nanoclustering following stimulation with EGF for 40 and 60 min (both P = 1).

FIG. 3.

Induction of EGFR nanoclustering following ligand binding requires RTK activity. (A) BHK cells were serum starved and then pretreated with 100 nM AG1478 for 10 min as indicated, followed by EGF stimulation in the presence or absence of AG1478 for 10 min as indicated. Whole-cell lysates were generated, and 20 μg of lysate was blotted for ppERK. Actin was used as a loading control. The graph represents the mean of three independent experiments (±SEM). Representative blots are shown. (B) BHK cells expressing EGFR-GFP were serum starved and treated with AG1478 for 10 min as indicated prior to stimulation with 50 ng/ml EGF for 10 min. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 13) standardized on the 99% CI. Significant differences from the control unstimulated EGFR pattern were assessed using bootstrap tests. Treatment with AG1478 led to a significant reduction in EGFR nanoclustering in the presence and absence of EGF (both P = 0.001).

FIG. 4.

PLD activity is elevated by EGF. BHK cells were serum starved and then stimulated with EGF for 1 min, and the production of phosphatidylbutanol was measured. Bars represent the relative proportion of phosphatidylbutanol to total lipid (n = 4) ± the standard deviation. Statistical significance was determined by t test (**, P = 0.002). The graph is representative of four independent experiments.

FIG. 5.

PLD2 activity is required for ligand-induced EGFR nanocluster formation. BHK cells expressing EGFR-GFP alone (B and C) or in combination with PLD2 K758R (A) were serum starved and treated as follows: (A) 50 ng/ml EGF for 10 min where indicated, (B) 200 μM propranolol for 30 min, followed by 50 ng/ml EGF for 10 min where indicated (C), or 50 ng/ml EGF for 10 min in the presence or absence of 1 μM R59949 where indicated. Plasma membrane sheets were generated and labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 6) standardized on the 99% CI. Significant differences from the control unstimulated EGFR pattern were assessed using bootstrap tests. (A) Expression of PLD2 K758R significantly decreased the basal level of EGFR nanoclustering (P = 0.001). (B) Treatment with 200 μM propranolol led to a significant increase in EGFR nanocluster formation (P = 0.001). (D and E) Whole lysates were extracted from cells treated as indicated for panels A and B, and 20 μg of each lysate was blotted for ppERK. Actin was used as a loading control. The graphs represent the mean of three independent experiments (±SEM). Significant differences from the control were assessed by t test. (D) The expression of PLD2 K758R led to a significant reduction in ppERK following EGF stimulation compared to those cells expressing EGFR alone (*, P = 0.02). (E) Treatment with 200 μM propranolol in the absence of EGF increased basal ppERK levels, although the increase did not reach significance (P = 0.2).

FIG. 6.

EGF stimulates PA production at the plasma membrane. (A) BHK cells expressing GFP NES Spo20p were serum starved and then stimulated with 50 ng/ml EGF. Cells were fixed and imaged by confocal microscopy. (B) Plasma membrane sheets were generated from BHK cells expressing GFP NES Spo20p that had been serum starved and then stimulated with EGF for the indicated times. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. (Left panel) The L(r) − r curve represents the clustering of GFP NES Spo20p after 10 min of EGF stimulation. (Right panel) The graph represents the mean number of gold particles/μm² (±SEM). (C) BHK cells expressing EGFR-GFP alone or in combination with mRFP NES Spo20p were serum starved and then stimulated with 50 ng/ml EGF for the indicated times. Cells were imaged in the frequency domain using a wide-field FLIM-FRET microscope. Data points represent the mean fluorescence lifetime of GFP (±SEM). The green dashed line represents the lifetime of EGFR-GFP in the absence of mRFP NES Spo20p. Representative fluorescence lifetime images are shown.

FIG. 7.

EGFR nanocluster formation is induced by treatment with exogenous PA but not PS. BHK cells expressing EGFR-GFP were serum starved and then stimulated with either (A) 100 μM PA or (B) 100 μM PS for the indicated times. Plasma membrane sheets were labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 14) standardized on the 99% CI. Significant differences from the control unstimulated EGFR pattern were assessed using bootstrap tests. (A) Treatment with PA led to a significant increase in EGFR nanoclustering at all time points (P = 0.001). (B) Incubation with PS did not have a significant impact on EGFR nanoclustering over time.

FIG. 8.

PA production regulates EGFR nanoclustering downstream of RTK activation. (A) BHK cells expressing EGFR-GFP were serum starved and treated with 100 nM AG1478 for 10 min as indicated prior to stimulation with 100 μM PA for 10 min. (B) BHK cells expressing EGFR-GFP in the presence or absence of PLD2 were serum starved and then stimulated with 50 ng/ml EGF for the indicated times. (C) BHK cells expressing EGFR-GFP were serum starved and then stimulated with 50 ng/ml EGF in combination with 100 μM PA for the indicated time. Plasma membrane sheets were generated and labeled with anti-GFP antibody conjugated to 5-nm gold particles. Univariate K functions are weighted means (n ≥ 9) standardized on the 99% CI. Significant differences from the control unstimulated EGF receptor pattern were assessed using bootstrap tests. (A) Treatment with AG1478 led to a significant reduction in EGFR nanoclustering (P = 0.041). However, cotreatment with PA reversed the effect of AG1478 treatment, leading to a level of EGFR nanoclustering that was not significantly different from that obtained by treatment with PA alone (P = 0.961).

FIG. 9.

PLD2 modulates EGF-induced signal transduction. BHK cells expressing EGFR-GFP in the presence or absence of PLD2 were serum starved and then stimulated with 50 ng/ml EGF for the indicated times. Whole-cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting for ppERK and α-tubulin. A representative blot is shown. Bars represent the mean fold increase in ppERK compared to control unstimulated cells (± SEM; n = 3).