The extracellular region of ErbB4 adopts a tethered conformation in the absence of ligand

Abstract

The human ErbB family of receptor tyrosine kinases comprises the epidermal growth factor receptor (EGFR/ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). ErbBs play fundamental roles in cell growth and differentiation events in embryonic and adult tissues, and inappropriate ErbB activity has been implicated in several human cancers. We report here the 2.4 A crystal structure of the extracellular region of human ErbB4 in the absence of ligand and show that it adopts a tethered conformation similar to inactive forms of ErbB1 and ErbB3. This structure completes the gallery of unliganded ErbB receptors and demonstrates that all human ligand-binding ErbBs adopt the autoinhibited conformation. We also show that the binding of neuregulin-1beta to ErbB4 and ErbB3 and the binding of betacellulin to both ErbB4 and ErbB1 does not decrease at low pH, unlike the binding of epidermal growth factor and transforming growth factor-alpha to ErbB1. These results indicate an important role for ligand in determining pH-dependent binding and may explain different responses observed when the same ErbB receptor is stimulated by different ligands.

Fig. 1.

Shared and specific ErbB ligands. (A) Overlapping specificities of ErbB ligands. Asterisks denote the more potent activators of ErbB4 signaling (36, 37). (B) Alignment of amino acid sequences of EGF-like domains in ErbB ligands. Dots indicate strictly conserved residues and histidines are in bold. Ligand numbering is consistent with previous structural reports (4, 5, 38).

Fig. 2.

Structure of sErbB4. (A) Ribbon diagram of sErbB4. Domains I, II, III, and IV are colored blue, green, yellow, and red, respectively. The N and C termini are indicated by the letters N and C. (B) Surface representation of sErbB4. The intramolecular contact between domains II and IV is boxed. (C) Domain II/IV contact in ligand-binding sErbBs. Residues at the tether between domains II and IV of sErbB4 are shown in green (domain II) and red (domain IV). The equivalent residues from tethered sErbB1 and sErbB3 are shown in gray and white, respectively (3, 6). The buried surface areas and the surface complementarity coefficients for each tether are indicated in the legend.

Fig. 3.

Comparison of ligand-binding surfaces in sErbB1 and sErbB4. (A) Conservation of sErbB1 TGF-α-binding residues in sErbB4. Residues with atoms within4Åofa TGF-α residue are shown in red on surface representations of sErbB1 domains I and III (4). Residues that are strictly conserved between the ErbB1 ligand-binding site and ErbB4 are colored blue on surface representations of sErbB4. (B) Electrostatic potential on the ligand-binding surfaces of ErbB1 and ErbB4. Regions with negative electrostatic potential are colored red and regions with positive electrostatic potential are colored blue (scale ± 10 e/kT).

Fig. 4.

Binding of sErbBs to their cognate ligands. (A) Binding of sErbB4 to BTC and NRG1β at pH 8.0. (B) Binding of sErbB1, sErbB3, and sErbB4 to immobilized ligands at different pH values. Experiments were performed by using surface plasmon resonance as described in Materials and Methods.