Ligand regulation of a constitutively dimeric EGF receptor

Abstract

Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

Figure 1. LET-23 is a ligand-regulated dimer.

(a) Domain compositions of human EGFR (hEGFR), C. elegans LET-23 and D. melanogaster EGFR (dEGFR), and members of the covalently dimerized IR family. Domains I and III (L1 and L2 in IR) are β-helix/solenoid domains. Domains II, IV and V (CR in IR) are cysteine rich. IR also has fibronectin type III (FNIII) domains. Intracellular tyrosine kinase (TK) domains are marked. Note the dimerization arm emanating from domain II in the EGFRs. (b) Size-exclusion chromatography–MALS (SEC-MALS) data for sLET-23ΔV (red) and human sEGFR (grey) at eluted concentrations of ∼1 μM. Circles denote molecular masses determined by in-line MALS (left axis). Including glycosylation, the monomeric molecular masses of sEGFR and sLET-23ΔV are both ∼80 kDa—with sEGFR running as a monomer and sLET-23ΔV as dimer. Retention volumes for protein molecular mass standards are marked (with kDa values) across the top of the figure. (c) Representative SE-AUC data for sLET-23 (8 μM, 8,200 r.p.m.). Without ligand (red circles), the data across all repeats fit well to a single ideal 190±22 kDa species—approximately twice the monomeric mass (∼91 kDa). In the presence of 60 μM LIN-3^EGF (blue circles), the fit is essentially unchanged, yielding a molecular mass of 185±6 kDa across all repeats (see Table 1). Fits to the data are shown as white curves superimposed on the data points and residuals are shown below the fits as open circles. (d) SAXS-derived normalized I(0)/c values for sEGFR and sLET-23ΔV, with and without bound ligand (added at 61–110 μM) at pH 8, using home-source data. The average I(0)/c for unliganded (monomeric) sEGFR was set to a relative value of 1.0 and all other I(0)/c values were normalized to this value, with mean±s.d. presented for 3–14 independent measurements. (e) Western blotting showing LIN-3^EGF-induced phosphorylation of full-length LET-23 in D. melanogaster S2 cells. The uncropped gel and molecular weight markers are shown in Supplementary Fig. 2.

Figure 2. Determinants of sLET-23 dimerization.

(a) Representative SE-AUC data for sLET-23 (red circles) and sLET-23^dim-arm (grey circles) collected at 5 μM and 10,000 r.p.m. (without added ligand), which fit to single ideal species of 190±22 and 94±7 kDa, respectively, across all repeats (see Table 1 and Supplementary Fig. 3). (b) Representative SE-AUC data for sLET-23ΔV (8 μM, 8,200 r.p.m.) with (blue circles) and without (red circles) 50 μM LIN-3^EGF. The data fit to single species of 141±3 and 153±27 kDa with and without ligand, respectively (see Table 1 and Supplementary Fig. 3), indicating that ligand binding does not alter oligomerization state. (c) Representative SE-AUC data for sLET-23ΔIV/V (8 μM, 8,200 r.p.m.) with 60 μM LIN-3^EGF (blue circles) or without ligand (red circles). The data show that sLET-23ΔIV/V is largely monomeric (82±8 kDa), and that ligand binding promotes dimerization (see Table 1 and Supplementary Fig. 3).

Figure 3. SAXS analysis of ligand-induced conformational changes in sLET-23ΔV dimers.

Experimental synchrotron scattering data for 42 μM sLET-23ΔV in the absence (a, red circles) or presence (b, blue circles) of 100 μM added LIN-3^EGF, collected at pH 8.0 at MacCHESS (see Supplementary Fig. 4). Data were acquired between 0.0164 Å⁻¹≤q≤0.456 Å⁻¹ and fit with GNOM (black line) as described in the Methods. Dilutions to 28 and 14 μM did not reveal spurious concentration-dependent effects. (c) SAXS-derived radial distance distributions (or P(r) curves) for 42 μM sLET-23ΔV with 100 μM LIN-3^EGF (blue points) or without added ligand (red points), calculated by inverse Fourier transforms of (a,b). SAXS-derived molecular envelopes, shown as grey mesh, for 42 μM sLET-23ΔV alone (d) or in the presence of 100 μM LIN-3^EGF (e). The EGF-stabilized dimer of human sEGFR from PDB entry 3NJP has been manually docked into both envelopes to illustrate the small scale of ligand-induced structural rearrangements. D_max (at 130±5 Å) is unchanged on LIN-3^EGF addition for these studies and the measured value for R_g is also unaltered, at 45.3±0.5 Å.