Development of ErbB4 antagonists
ErbB4 is a member of the ErbB family of receptor tyrosine kinases. Because of a paucity of appropriate pharmacologic tools, little is known about the roles ErbB4 plays in mammalian development and tumorigenesis. In response to this need, we hypothesized that a recombinant form of the extracellular domain of ErbB4 would antagonize ligand-induced ErbB4 tyrosine phosphorylation and subsequent downstream signaling and could be used to probe ErbB4 function. Indeed, we show here that one such ErbB4 protein, secErbB4-26/549, is a potent inhibitor of ligand-induced ErbB4 tyrosine phosphorylation and of ligand-induced ErbB4 coupling to biological responses. Furthermore, we demonstrate that secErbB4-26/549 antagonizes ligand-induced ErbB4 signaling by acting as a ligand sink. Unfortunately, there are limitations to secErbB4-26/549. In general, more than one ErbB family member is expressed by a given cell and many ErbB4 ligands can bind other ErbB receptors. Moreover, ErbB4 heterodimerizes with other ErbB family members and that may be a relevant physiological outcome of ligand binding to ErbB4. Therefore we still wanted to develop novel competitive antagonists for ErbB4. Thus, we need to first identify where natural ErbB4 agonist bind the receptor. We hypothesized that Leu437 and Lys438 of ErbB4 interact with Phe45 of the ErbB4 agonist NRG2β. Consequently, we predicted that substituting this putative ligand-binding motif of ErbB4 (Leu437 and Lys438) for the corresponding residues of the EGFR (Val441 and Ser 442) would generate a gain-of-function EGFR mutant that exhibits increased affinity for ErbB4 ligands. We also hypothesized that hydrophobicity in the putative ligand binding site of ErbB4 regulates ligand binding. Thus, we have generated and analyzed four EGFR mutants (V441L, S442K, S442F and S442L). In this work we demonstrate that NRG2β is a more potent agonist and displays higher affinity for one of the EGFR mutants, EGFR-S442F, that the wild-type EGFR. Furthermore, the EGFR-S442F mutant is coupled to ligand-independent suppression of apoptosis via PI3K and Akt. We will place this EGFR mutant in the context of an emergent model that describes the role of EGFR in tumorigenesis.
Riese, Purdue University.
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