An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family

Abstract

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.

Figure 1.

Model of ErbB activation. (A) ErbB1 in a quiescent cell (no EGF, low free [Ca²⁺] in cytosol). The cytoplasmic leaflet of the plasma membrane contains acidic phospholipids (lipids with red minus signs) that strongly attract the reversible membrane anchor region of the JM domain, comprising eight basic (circled blue plus signs) and five hydrophobic residues (green circles). The PTK core (depicted with the COOH-terminal portion of the JM domain as a rectangle of appropriate dimensions, 5 × 6 nm) has a positively charged face (blue plus signs) that also binds electrostatically to the bilayer. We postulate that these membrane interactions inhibit the enzyme. The length of the first two portions of the JM domain, as well as the size of the PTK domain and calcium/calmodulin (Ca/CaM), are drawn approximately to scale with the bilayer (thickness ∼5 nm). (B) Activated ErbB1. After ligand-induced dimerization (not depicted) and partial trans autophosphorylation, we postulate that binding of acidic Ca/CaM (charge = −16) to the basic (charge = +8) reversible membrane anchor forms a high affinity complex with a net charge of –8; this charge reversal repels the complex from the negatively charged bilayer, ripping both the JM and PTK domains off the membrane and fully activating the ErbB1. The PTK core is now free to rotate via the flexible linker, giving the catalytic site access to tyrosines on an adjacent ErbB family member (not depicted), or other substrate. As discussed in the text, Ca/CaM may cycle on and off the JM region at up to a diffusion limited rate (∼10² s⁻¹ if [Ca/CaM] = 10⁻⁶ M). (C) Sequence of the JM region of ErbB1. SwissProt accession no. P00533.

Figure 2.

Electrostatic potential profile adjacent to the ErbB1 PTK core. The red and blue meshes illustrate the −25 and +25 mV equipotential profiles, respectively. Potentials calculated from the Poisson-Boltzmann equation in 100 mM salt and illustrated using GRASP. (A) ErbB1 PTK (together with residues 673–682 of the JM) domain, as revealed by the crystal structure (Stamos et al., 2002). The orientation is the same as depicted in Fig. 1 A; the membrane is above the basic (blue) face. (B) Structure rotated 90° to show the positively charged face. Residues 673–682 are the extended region at the top of the structure, starting from N.

Figure 3.

Binding of ErbB1(645–660) to phospholipid vesicles. Vesicles were formed from a mixture of the zwitterionic lipid PC and the acidic lipid PS. 100 nm LUVS were formed from 2:1 PC/PS (filled circles) or 5:1 PC/PS (open circles); the external aqueous solutions contained 100 mM KCl, 1 mM MOPS, pH 7.0. The binding measurements were made with a low (∼4 nM) concentration of radiolabeled (NEM) peptide using a centrifugation assay. The solid lines through the points illustrate the fit of Eq. 1 to the data. The molar partition coefficient, K (or apparent association constant of the peptide with a lipid), is the reciprocal of the lipid concentration that binds 50% of the peptide; the dotted line indicates K ≅ 2 × 10⁴ M⁻¹ for 5:1 PC/PS vesicles. The bars through the data points illustrate the SD of n = 4 independent experiments.

Figure 4.

Ca/CaM binds with high affinity to ErbB1(645–660) and prevents its association with lipid vesicles. The percent bound peptide, present only at a trace concentration (∼5 nM), is plotted as a function of the concentration of CaM in the presence (open circles, [Ca²⁺]_free ∼20 μM) or absence (filled circles) of Ca²⁺. The total lipid concentration is 2 × 10⁻⁵ M and the solutions contain 100 mM KCl, 1 mM MOPS, pH 7.0, 100 μM EGTA, ±120 μM CaCl₂. The solid curve illustrates the prediction of Eq. 2, taking the association constant of the peptide with the membrane, K = 10⁶ M⁻¹ (Fig. 3), and deducing the association constant of the peptide with Ca/CaM, K_CaM = 10⁸ M⁻¹ from the fit of Eq. 2 to the data. The fraction of radioactively labeled peptide bound to the 100 nm 2:1 PC/PS large unilamellar vesicles (LUVs) was determined by a centrifugation technique.

Figure 5.

The calmodulin inhibitor W7 inhibits the EGF-mediated autophosphorylation of the ErbB1 receptor. Cos1 cells were treated with 20–50 μM W-7 for 30 min, and then exposed to 100 ng/ml (∼20 nM) EGF for 10 min. The cells were lysed and treated with rabbit anti-ErbB1 to immune precipitate the receptor. Western blots were performed on the immunoprecipitates using the murine antiphosphotyrosine monoclonal antibody 4G10, (top panel). Reprobing of the same blot with a rat anti-ErbB1 monoclonal antibody (bottom panel) demonstrated that W-7 treatment did not affect receptor levels during the time frame of the experiment.