Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10 5 − 10 6 receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.EGFR | epidermal growth factor | ErbB proteins | receptor clusters | signal transduction E rbB proteins (ErbB1-4, HER1-4) constitute the best characterized family of receptor tyrosine kinases (1). Biochemical analysis has demonstrated that ErbB1, the prototypical member of the family (also known as the epidermal growth factor receptor, EGFR or HER1) undergoes ligand-induced homodimerization as a key step in its activation (2). More recent crystallographic studies reveal that ligand binding induces a transition from a closed conformation of ErbB1 to an extended configuration with the capacity to dimerize via intermolecular interactions mediated by domain II (3, 4). The ultrastructural data also confirm that dimerization of ErbB1 plays a fundamental role in activating the kinase domain by a mechanism resembling that of cyclin dependent kinases (5, 6). The coreceptor ErbB2 has no known ligand but upon transactivation expresses the most potent kinase activity of the ErbB family, thereby increasing the efficiency of signaling mediated by ErbB2-containing heterodimers (7). ErbB2 constitutively adopts an extended conformation potentiating the formation of heterodimers (8, 9) that can be inhibited by pertuzumab, a monoclonal antibody sterically blocking the heterodimerization arm of ErbB2 (10). Although the extracellular domain of ErbB2 has failed to form crystallographic homodimers, molecular biological and fluorescence resonance energy transfer (FRET) experiments have shown that full-length ErbB2 exists in dimers or higher-order aggregates in the plasma membrane (11,12). The implication is that the transmembrane, juxtamembrane and other intracellular domains (5, 13, 14) act in conjunction with the membrane environment (15) to mediate the dimerization and, thereby, functional states of ErbB proteins.Many investigators have sought to determine the distribution of ErbB1 in ...
While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance.
Specific inhibition of members of the EGFR (epidermal growth factor receptor) family, particularly EGFR and HER2 (human epidermal growth factor receptor 2), are an important therapeutic strategy in many human cancers. Compared with classical chemotherapy, these targeted therapeutics are very specific and initially effective, but acquired resistance against these targeted therapies is a recurring threat. A growing body of recent work has highlighted a pseudokinase in the EGFR family, HER3, and its ligand, NRG (neuregulin β1), to be of importance in models of resistant cancers, as well as in patients. In the present article, we describe some of the roles in which HER3 can mediate acquired resistance and discuss the current efforts to target HER3 itself in cancer.
Pseudokinases, the catalytically impaired component of the kinome, have recently been found to share more properties with active kinases than previously thought. In many pseudokinases, ATP binding and even some activity is preserved, highlighting these proteins as potential drug targets. In both active kinases and pseudokinases, binding of ATP or drugs in the nucleotide-binding pocket can stabilize specific conformations required for activity and protein-protein interactions. We discuss the implications of locking particular conformations in a selection of (pseudo)kinases and the dual potential impact on the druggability of these proteins.
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