Discovery and Characterization of Ligands for the Receptor Tyrosine Kinase ALK: AUG-alpha, AUG-beta and Heparin

Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. Ligand-mediated activation of RTKs is important throughout development and in disease states such as cancer. Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" RTKs in which the ligand(s) are unknown. ALK is particularly relevant to the development of the nervous system and aberrant activation of ALK drives subsets of lung adenocarcinoma, neuroblastoma, and other cancers. In this dissertation, I present three novel ligands for ALK: heparin, Augmentor-alpha (AUG-alpha), and Augmentor-(3 (AUG-beta). Heparin binds specifically to the ALK extracellular domain and activates ALK in a heparin chain length dependent manner. Heparins with short chain lengths bind to ALK in a monovalent manner and do not activate the receptor, whereas longer heparin chains induce ALK dimerization and activation in cultured neuroblastoma cells. Moreover, antibodies that bind to the extracellular domain of ALK interfere with heparin binding and prevent heparin-mediated activation of ALK. Thus, heparin and related glycosaminoglycans function as ligands for ALK. The ALK ligand AUG-alpha binds with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK, or the related receptor LTK, demonstrate that AUG-alpha binds and robustly activates both ALK and LTK. The previously established LTK ligand, AUG-beta, is specific for LTK and weakly binds to ALK. Furthermore, expression of AUG-alpha stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer driven by ALK. These experiments reveal the hierarchy and specificity of these two novel cytokines as ligands for ALK and LTK, along with heparin, and set the stage for elucidating their roles in development and disease states.