The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva TM ). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.Growth factor interactions with cell surface receptors influence proliferation, survival, differentiation, and metabolism (1). The loss of control over these vital cellular processes is a hallmark of oncogenesis (2). For instance, aberrant signaling from overexpressed growth factor receptor ErbB2 is causal in approximately 30% of invasive breast cancers (3). Growth factors bind to a cognate membrane-bound receptor system and mediate changes in the intracellular portion of the receptor, often through the formation of dimers or oligomers of receptors that initiate signal transduction cascades. The epidermal growth factor receptor (EGFR, 1 also ErbB1 or HER1) and its ligands, epidermal growth factor (EGF) and transforming growth factor-␣, are among the earliest characterized members of the growth factor/receptor tyrosine kinase (RTK) family. In contrast to the widely applicable ligand-induced receptor dimerization paradigm, there is evidence that EGFR family members exist as preformed dimers (4) and form higher oligomer signaling complexes (5). Normal signaling in the EGFR system involves ligand-induced homo-oligomerization or hetero-oligomerization with the closely related RTKs ErbB2 (HER2), ErbB3 (HER3) and/or ErbB4 (HER4) (6). Autophosphorylation of key tyrosine residues within the carboxyl-terminal portion of the receptor provides sites for direct interaction with SH2-containing proteins, leading to subsequent signal transduction events.The EGFR system, including receptor homologues and relevant ligands, is complex. There are at least 12 different ligands that bind to the EGF receptor family with partially redundant specificity for certain receptors. Several of the ligands including EGF, transforming growth factor-␣, heparin-binding EGF, and betacellulin are reported to bind to EGFR with nanomolar dissociation constants (7). Betacellulin also binds ErbB4 with high affinity. Similarly, heregulin binds to ErbB3 or ErbB4 with dissociation constants in the nanomolar range. So ...

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.

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ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.