The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and… (More)
FIGURE 2. Interaction specificity analysis of peptide aptamers with intracellular growth factor receptor domains in vivo ; yeast two-hybrid experiments. The KF1 cells were cotransformed with bait constructs encoding the complete kinase domain of ErbB2 (ErbB2-KDg), IGF-IR (IGF-IR-KDg), or EGFR (EGFRKDg) and with aptamer encoding prey constructs [Ag-11, AII-7, and thioredoxin (Trx)] to determine the interaction specificity of individual aptamers. The receptor domain encoding constructs comprised a Gal4-DBD and the aptamers or empty thioredoxin fused to the Gal4-AD. The transformed yeast cells were grown overnight in liquid cultures and plated the next day on media lacking leucin and tryptophan ( LT ) as control for transformation of both vectors and on media lacking leucin, tryptophan, and histidine ( LTH ). For plating, the A600 of the culture was adjusted to 0.7 from which 1:10 serial dilutions were made. The KF1 yeast cells are only able to synthesize histidine on intracellular interaction of the receptor domain and peptide aptamer, which in turn allows growth under selective conditions ( LTH). The construct encoding the thioredoxin scaffold protein without a peptide sequence served as a control and does not interact with ErbB2-KDg (lane 1). Ag-11 interacts with the ErbB2-KDg domain (lane 2 ) but not with the EGFR-KDg (lane 3 ). AII-7 interacts with ErbB2-KDII (lane 4) but not with the kinase domain of IGF-IR (lane 5) and only slightly with the EGFR-KDg (lane 4).