Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes, such as cell cycle, differentiation, proliferation, apoptosis and survival. Mutations, hyperactivity and loss of function of RTKs are responsible for numerous diseases. Because of the therapeutic importance of RTK signaling, intensive studies have been devoted to understanding the signaling mechanisms of RTKs, and the key components in their signaling networks. However, studying the cellular responses to RTK stimulation in a native cellular context is technically challenging. Consequently, many details of RTK signaling kinetics, and the underlying molecular mechanisms of action, remain unclear. The RET receptor tyrosine kinase is important for neuronal cell survival and function, and for the development of the kidneys and nervous system. Gain of function of RET leads to tumor formation, while loss of function in RET’s kinase activity is associated with the developmental kidney defect Hirschsprung’s disease. RET is activated by ligands of glial cell line-derived neurotrophic factor (GDNF) family, which consist of four homologs—GDNF, Neuturin, Artemin (ART) and Persephin. GDNF family ligands activate RET only in the presence of GPI-linked co-receptors (GFRα1–4). Formation of the pentameric ligand/co-receptor2/RET2 complex leads to dimerization of RET and autophosphrylation of its cytoplasmic kinase domain. RET phosphorylation results in the activation of multiple downstream signaling pathways, including the Ras-Raf-MEK-ERK and PI3K-Akt pathways. The ERK and Akt signaling pathways participate in a variety of cellular activities, including cell survival, proliferation, and differentiation. My project addresses the following questions: (1) What are the kinetics of RET activation and deactivation processes after ART stimulation? (2) How is RET activation coupled to the phosphorylation of ERK1/2 and Akt? (3) How does ligand-induced internalization of RET affect RET signaling? (4) How does each step in the RET-Ras-Raf-MEK-ERK cascade quantitatively regulate ERK phosphorylation levels? The results will elucidate the spatial and temporal dynamics of RET signaling upon stimulation by ART, and to determine how downstream signaling is regulated by the amplitude and timing of RET activation. Overall, the thesis aims to advance our understanding of RTK signaling, by establishing methods and principles that can potentially be applied to other RTK systems.

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are peptides implicated in the inflammatory response. They are released in increased amounts during inflammation and induce thermal hyperalgesia. Whether these molecules directly affect the sensitivity of primary nociceptive sensory neurons is unknown. This information could provide a link between increased inflammation-induced release of GFLs and their ability to promote inflammatory hyperalgesia. These molecules bind to one of four GFRα receptor subtypes, and this GFL-GFRα complex often translocates to the receptor tyrosine kinase, Ret. The focus of this dissertation was to determine whether GFLs modulate the stimulated-release of calcitonin gene-related peptide (CGRP). Isolated sensory neurons and freshly dissociated spinal cord tissue were used to examine the enhancement in stimulated-release of CGRP, a measure of sensitization. Exposure of isolated sensory neurons to GDNF, neurturin, and artemin, enhanced the capsaicin stimulated-release of immunoreactive CGRP (iCGRP). Sensitization by GFLs occurred in freshly dissociated spinal cord tissue. Persephin, another member of the GFL family, did not enhance stimulated-release of iCGRP. These results demonstrate that specific GFLs are mediators of neuronal sensitivity. The intracellular signaling pathways responsible for this sensitization were also evaluated. Inhibition of the mitogen activated protein kinase (MAPK)/extracellular signal-related kinase 1/2 (Erk 1/2) pathway selectively abolished the enhancement of CGRP release by GDNF. NTN-induced sensitization was abolished by inhibition of the phosphatidylinositol-3-kinase (PI-3K) pathway. Reduction in Ret abolished the GDNF-induced sensitization, but did not fully inhibit NTN or ART-induced sensitization. Inhibition of other cell surface receptors (neural cell adhesion molecule (NCAM), and Integrin β-1) had distinct effects on the sensitization capability of each of the GFLs. Ret and NCAM inhibition in combination abolished ART-induced sensitization. It was necessary to inhibit Ret, NCAM, and Integrin β-1 to prevent the NTN-induced sensitization. These data demonstrate that the GFLs use distinct signaling mechanisms to induce the sensitization of nociceptive sensory neurons. The work presented in this thesis provides the first evidence for these novel and distinct Ret-independent pathways for GFL-induced actions and provides insight into the mechanism of sensory neuronal sensitization in general.