U.S.A. these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors. (15) has suggested that the phosphorylation of ErbB2 occurs within ErbB2/ErbB3 tetramers in which the active ErbB2 from one dimer phosphorylates the ErbB2 in the other dimer. Thus, signaling by ErbB2/ErbB3 heterodimers necessitates the formation of higher-order receptor oligomers not required by other ErbB pairings. In previous work we have used luciferase fragment complementation imaging to characterize the interaction Solithromycin of the EGF receptor with itself (16, 17) as well as with ErbB2 (18, 19). In this work, we extended these studies to include a characterization of dimers containing ErbB3. We also investigated the effect of ErbB-targeted drugs on these ErbB receptor Solithromycin dimers. Our data demonstrate that ErbB3 interacts with itself as well as with the EGF receptor and ErbB2. These interactions are differentially affected by EGF and NRG-1 as well as the ErbB-targeted therapeutics. Although catalytically impaired, ErbB3 binds both erlotinib and lapatinib, and this alters its ability to interact with other ErbB receptors. These data clarify the hierarchy of interactions among the ErbB receptors and suggest that small-molecule tyrosine kinase inhibitors likely alter the dynamic equilibrium between ErbB3 and other ErbB family members. EXPERIMENTAL PROCEDURES Materials EGF was purchased from Biomedical Technologies. NRG-1 was purchased from R&D Systems. Antibodies to the EGFR, EGFR pTyr-1068, ErbB2 pTyr-1221/1222, and ErbB3 pTyr-1289 were purchased from Cell Signaling Technology. Antibodies against ErbB2 and ErbB3 were purchased from Millipore. Cetuximab was obtained from the Barnes Hospital pharmacy. Trastuzumab (HerceptinTM) and pertuzumab (PerjetaTM) were provided by Genentech (South San Francisco, CA). Erlotinib was from Axon Medchem. Lapatinib was chemically synthesized and was a gift from Ron Bose Fst (Washington University). A30 was generated enzymatically and purified as described previously (20). FetalPlex was from Gemini Bio-Products. ErbB3 in pcDNA3.1+ was a gift from Mark Lemmon (University of Pennsylvania). DNA Constructs The EGF receptor and ErbB2 fused to a flexible linker and either the N-terminal fragment of luciferase (NLuc) or the C-terminal fragment of luciferase (CLuc) in pBI-Tet or pcDNA3.1 were generated as described previously (18). In these constructs, the ErbB receptor can be removed by digestion with NheI and BsiWI. This leaves the BsiWI-to-NheI vector fragment with the flexible linker plus the luciferase fragment. The ErbB3-NLuc and ErbB3-CLuc constructs were generated by introducing an upstream NheI site via QuikChange mutagenesis into ErbB3 in pcDNA3.1+. A BsiWI site was then engineered at the C terminus of ErbB3, removing the stop codon. The resulting ErbB3 insert was then isolated by digestion with NheI and BsiWI and ligated with the BsiWI-to-NheI vector portion of pBI-Tet or pcDNA3.1 containing the NLuc or CLuc fragment. Cell Lines A stable line of CHO cells expressing EGFR-NLuc on Solithromycin a Tet-inducible plasmid was transfected with ErbB3-CLuc in pcDNA3.1-Zeo. EGFR-NLuc/ErbB3-CLuc clones were selected by growth in 500 g/ml zeocin (Invitrogen). Similarly, a stable line of CHO cells expressing Tet-inducible ErbB2-NLuc was transfected with ErbB3-CLuc in pcDNA3.1Zeo. ErbB2-NLuc/ErbB3-CLuc clones were selected by growth in 500 g/ml zeocin. A double-stable line of CHO cells expressing ErbB3-NLuc and ErbB3-CLuc was isolated by first transfecting parental Tet-on CHO cells with ErbB3-NLuc in pBI-Tet plus pTK-Hyg and selecting stable clones by growth in 500 g/ml hygromycin (Invitrogen). A single stable ErbB3-NLuc line was then transfected with ErbB3-CLuc in pcDNA3.1Zeo, and ErbB3-NLuc/ErbB3-CLuc clones were selected by growth in 500 g/ml zeocin. Luciferase Assays Cells were plated into 96-well black-walled dishes 48 h prior to use and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FetalPlex and the desired concentration of doxycycline (Clontech). Immediately prior to the assay, cells were transferred into Dulbecco’s modified Eagle’s medium without phenol red with.