Regulation of human EGF receptor by lipids

Abstract

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.

Fig. 1.

Full-length EGF receptor reconstituted in proteoliposomes shows only low affinity binding of EGF. Representative saturation plots for ld (A) and ld/lo proteoliposomes (B) before background subtraction. Squares show background binding to liposomes. The assayed ligand concentration range of the respective measurement was between 24 pM to 97 nM. Insets represent Scatchard transformation of the respective saturation binding curves after background subtraction. The measured K_D values for EGF binding were in the range of 2–5 nM for all lipid compositions (n > 5) (see SI Text), corresponding to low affinity state EGF binding. Saturation binding data was analyzed by nonlinear curve fitting using GraphPad Prism 5.0 software.

Fig. 2.

GM3 inhibits activity of EGF receptor in ld/lo proteoliposomes. The activity of EGF receptor reconstituted in various lipid environments was measured by antibody detection of transphosphorylated Y1173. Proteoliposomes were incubated in the presence of ligand (0.75 and 150 ng/mL) and ATP (0.25 mM). The activity of the kinase domain was measured by detection of the phosphorylation of Y1173 by two antibodies mapping the same residue in either phospho- or dephospho- state. Representative blots for phosphorylation of EGF receptor are shown for ld (A) or ld/lo (B) liposomes. (C) The activity of the receptor in ld proteoliposomes is not affected by addition of glycolipids, whereas (D) addition of 0.5 mol% GM3 in ld/lo proteoliposomes leads to total inactivation of EGF receptor activity. Other glycolipids had no effect. Error bars represent SD values. GM3-ld/lo phosphorylation was assayed with ten independent proteoliposomes preparations. All other conditioned were assayed n > 3. Representative Western blots of all lipid compositions can be seen in SI Text. Western blots were analyzed using AIDA software (Raytest).

Fig. 3.

Neuraminidase treatment of GM3-ld/lo proteoliposomes rescues EGF receptor autophosphorylation. (A) The ld/lo proteoliposomes containing GM3 were incubated with neuraminidase (30 min, 37 °C), which hydrolyses NeuAc from the headgroup of GM3 converting it to Lac-Cer. (B) HI (heat inactivated) neuraminidase has no effect on the inhibitory effect of GM3 in ld/lo proteoliposomes. The presence of active neuraminidase results in full rescue of the WT-EGFR autophosphorylation. In total three independent experiments have been performed.

Fig. 4.

Mutation of membrane proximal K642 leads to GM3 insensitivity. K_D values of EGFR-K642G mutant receptor ld/lo ± GM3 proteoliposomes is not affected to a great extent compared to WT-EGFR (SI Text). Displayed K_D values correspond to a same proteoliposome batch used for the autophosphorylation assay shown in (B). Error bars correspond to the standard error of the mean of duplicate saturation binding measurements. Three independent ligand-binding assays have been performed in total. (B) EGFR-K642G mutation results in an insensitivity with respect to the inhibitory effect of GM3 on EGFR autophosphorylation.

Fig. 5.

Ligand-induced dimerization of EGF receptor in proteoliposomes. Prior to cross-linking, EGFR proteoliposomes were incubated with EGF (30 min, RT). For chemical cross-linking BS³ was used (50 μM, 15 min, RT). In the absence of ligand, a significant fraction of the EGFR appears as preformed dimers. In ld/lo proteoliposomes the presence of GM3 is accompanied by a significant decrease of this preformed dimer fraction whereas ligand driven dimerization is not affected. Molecular weight determination is shown in SI Text. In ld proteoliposomes GM3 has no effect in EGFR dimer formation. In total four independent experiments have been performed.