General and Specific Receptor Tyrosine Kinase Signaling Mechanisms Revealed by Analysis of Chimeric EGFR and IR Family Receptors
McCabe, Jacqueline M.
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Receptors embedded in the plasma membrane serve to relay signals from the outside to the inside of the cell and regulate cell growth and differentiation. Disruption of these signals can occur by mutation or abnormal expression of receptor proteins. These disruptions can result in aberrant cell growth and are found in many cancers. As a result, cell surface receptors are common targets of directed small molecule and biologic therapeutics. Two such targeted receptors are Smoothened (Smo) and the Epidermal Growth Factor Receptor (EGFR). Mutations in Smo are found in basal cell carcinoma, meningiomas and medulloblastoma. Overexpression or mutations of EGFR are found in glioblastoma and non-small cell lung cancer. Approved therapeutics targeting Smo and EGFR are currently used in the clinic, but acquired resistance to both Smo and EGFR targeted therapeutics generally occurs within months or at most a few years. More precisely defining the molecular mechanisms of activation and inhibition of these two receptors is of both fundamental interest as well as critical to understanding and improving current targeted therapeutics. Ligand-induced dimerization of receptor tyrosine kinases (RTKs) is a generally accepted mechanism of activation for all classes of RTKs except the Insulin Receptor (IR) family. In contrast to all other RTK families, IR family members exist as preformed disulfide linked dimers on the cell surface. Recent evidence suggests that the ectodomain (ECD) of IR, and its homolog the type 1 insulin-like growth factor receptor (IGF1R), maintains separation of subunit transmembrane regions in the absence of ligand. Ligand binding releases this ectodomain autoinhibition and allows the transmembrane domains to come together and the intracellular kinase domains to transphosphorylate. Parallels between the IR/IGF1R mechanism of activation and ligand-induced dimerization suggested that the IR/IGF1R ECDs may be able to regulate other families of RTKs, and we set out to understand how the IR/IGF1R activation mechanism relates to EGFR activation. We find that the IR/IGF1R ECD fails to activate the kinase domain of EGFR in either IR-EGFR or IGF1R-EGFR chimeric receptors. In contrast, the EGFR ECD is able to activate the IR kinase domain in an EGFR-IR fusion in an EGF-dependent fashion. The IR and EGFR mechanisms of activation thus appear to share some but not all features. Despite years of intensive study and its central importance to animal development and human health, our understanding of the Hedgehog (Hh) signaling pathway remains riddled with gaps, question marks, assumptions, and poorly understood connections. In particular, understanding how interactions between Hh and Patched (Ptc), a 12-pass integral membrane protein, lead to modulation of the function of Smo, a 7-pass integral membrane protein, has defied standard biochemical characterization. Recent structural and biochemical characterizations of Smoothened domains have begun to unlock this riddle, however, and point towards improved cancer therapies. We present purification and biochemical characterization of near full-length Smo protein in the presence of small molecules targeting multiple Smo domains. These studies were carried out with the aim of developing the tools necessary to understand the molecular mechanisms governing Smo activity.