OPCML directly interacts with the extracellular domain of EPHA2, FGFR1, and HER2, a prerequisite for downregulation. OPCML directly interacts with the extracellular domain of EPHA2, FGFR1, and HER2, a prerequisite for downregulation. A, immunoprecipitation for OPCML showing binding for EPHA2, FGFR1, and HER2. Similarly, immunoprecipitation for each of these proteins in turn showed reciprocal binding for OPCML. We were not able to demonstrate coimmunoprecipitation with EGFR and OPCML after numerous attempts. B, GST pull-down assay with SKOV-3 cell lysate showing binding of HER2 and FGFR1 to GST-OPCML 1–3 and no binding of EGFR to GST-OPCML 1–3 protein. The presence of OPCML fusion proteins in these assays was verified by immunoblotting (bottom). C, confirmatory in vitro interaction studies were undertaken verifying binding of HER2 and FGFR1 extracellular domain protein, generated from in vitro translation kit (TnT; HER2 extracellular domain) and in Escherichia coli (FGF receptor-extracellular domain) to GST-OPCML 1–3 fusion protein. Both HER2 and FGFR1 extracellular domains were seen to bind to GST-OPCML 1–3. D, to demonstrate a mechanistic link between the RTK extracellular domain and OPCML, we undertook transient transfection of His-tagged full-length Neu/HER2 extracellular domain or extracellular domain-less Neu (Δ5-P95), and we demonstrated downregulation of the transfected full-length 185-kD extracellular domain containing the Neu in either the OPCML BKS2.1 or OPCML transiently cotransfected Cos-1 (Cos-Neu + OPCML); in contrast, there was no change in expression of the transiently transfected extracellular domain-less Neu (Δ5-P95) in the presence of OPCML. E, the relative mean band intensity from 5 separate Neu construct transfections in BKS2.1/SKOBS-V1.2 and 3 separate cotransfections in Cos1 cells shows that in both cell systems, the level of full-length Neu protein is significantly reduced by 50% to 80% when coexpressed with OPCML (P = 0.009 and P = 0.041, respectively), whereas the level of the extracellular domain-less 95-kD Neu species is unchanged in both systems regardless of OPCML status. F, growth assays in Neu and OPCML nonexpresing Cos-1 cells conclusively demonstrate that the significant growth acceleration (P = 0.003) induced by transiently transfected p185 Neu (Cos+ Neu) is abrogated by transient cotransfection (dotted line) with OPCML (Cos + Neu + OP, *P = 0.004 at 48 hours, **P = 0.016 at 72 hours), reducing the growth rate to that of the empty vector (Cos + EV) and OPCML (Cos + OPCML) controls. In contrast, the similarly significantly growth accelerated extracellular domain-less Δ5-P95 (P = 0.015) was not inhibited by OPCML cotransfection (Cos + Neu + OP, ***P = 0.56 at 48 hours, ****P = 0.092 at 72 hours), and this demonstrates the functional necessity of the extracellular domain for regulation of the RTK by OPCML. IB, immunoblotting; IP, immunoprecipitation. Arthur B. McKie et al. Cancer Discovery 2012;2:156-171 ©2012 by American Association for Cancer Research