Mechanism of activation and inhibition of the HER4/ErbB4 kinase

Abstract

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

Figure 1

Ribbon diagrams of ErbB4 kinase structures. (A) The active conformation of the ErbB4 kinase. The activation loop is colored red. (B) An inactive conformation of the ErbB4 kinase in complex with lapatinib, which is shown as a red stick model. The activation loop is disordered, but the loop termini are colored red. (C) Superposition of the active and inactive conformations of the ErbB4 kinase.

Figure 2

Superposition of the asymmetric dimers of EGFR and ErbB4 kinase. The ErbB4 kinase subunits are colored blue and cyan. An EGFR dimer is colored light yellow. Only the single blue colored ErbB4 kinase subunit and corresponding EGFR subunit were included in the superposition.

Figure 3

Receptor activation assay. (A) Western blot analysis of the indicated ErbB4 variants immunoprecipitated from cell lysates following treatment with or without Neuregulin-2β (NRG-2β). Blots were probed with an anti-phosphotyrosine antibody (top) or anti-ErbB4 antibody (bottom). (B) Quantitation of Western blot band intensities. The results are the mean and standard deviation from three independent experiments.

Figure 4

Bound lapatinib. (A) The 2F_o-F_c simulated annealing omit map for lapatinib contoured at 1σ. The structure of ErbB4 kinase is colored yellow, and lapatinib is shown as a red stick model. The structure of the EGFR kinase-lapatinib complex is superimposed with the EGFR kinase colored pale cyan and lapatinib green. (B) Chemical structure of lapatinib.