DISTINCT FUNCTIONS OF THE ALTERNATIVELY SPLICED ISOFORMS OF GDNF RECEPTOR COMPLEXES

Abstract

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a family of ligands (GFLs) that are known to be involved in many aspects of neurobiology, including neuronal differentiation, proliferation, survival and migration. GFLs activate downstream signaling through a multi-component receptor complex consisting of a preferred high-affinity GDNF family receptor alpha (GFRa) and co-receptors including Ret and/or neural cell adhesion molecule (NCAM). GFRa1 and GFRa2 have been identified as the preferred receptors for GDNF and NTN, respectively. Multiple alternatively spliced isoforms of the GFRa1, GFRa2 and Ret have been identified in the nervous system, but whether these isoforms serve distinct or redundant functions are not well addressed. The studies in this thesis are focused on unraveling the distinct biochemical and phenotypical effects of ligand activated GFRa1, GFRa2 and Ret receptor isoforms on neuronal cell differentiation and glioma cell migration. GFRa1 is alternatively spliced into two isoforms: GFRa1a and GFRa1b. Upon ligand stimulations, GFRa1a induced neurite outgrowth in neuroblastoma cells via ERK1/2 and Rac1/Cdc42 activations. On the contrary, GFRa1b inhibited GFRa1a-mediated neurite outgrowth through the activation of RhoA-ROCK pathway. Compared to normal brain which expressed comparable levels of GFRa1 isoforms, human glioma specimens were found to predominately express GFRa1b. Interestingly, GFRa1b but not GFRa1a was required for GDNF-induced RhoA expression and migration of C6 glioma cells. These findings suggest the differential involvement of GFRa1 isoforms in neuronal differentiation and glioma motility. Alternative splicing of GFRa2 results in at least three isoforms: GFRa2a, GFRa2b and GFRa2c. Upon ligand stimulations, GFRa2a and GFRa2c but not GFRa2b promoted CREB phosphorylation, sustained ERK1/2 activation and neurite outgrowth in PC12 cells. Interestingly, agonists of cAMP-PKA pathway cooperated with NTN to promote sustained ERK1/2 activation and neurite outgrowth through GFRa2b. Furthermore, cAMP-PKA signaling was found to be involved in GFRa2a and GFRa2c mediated neurite outgrowth. This study has not only identified the involvement of cAMP-PKA signaling in GFLs-induced neurite outgrowth but also provided novel insights into the functional difference of GFRa2 isoforms. Two major Ret isoforms, Ret9 and Ret51, produced by alternative splicing at C-termini have been identified. GDNF signaling via GFRa1a was able to induce neuronal differentiation through both Ret isoforms. Intriguingly, Ret9 but not Ret51 regulated activation of FRS2a and Src. Ret9-mediated late ERK1/2 activation and neurite outgrowth were dependent on Src kinase activity. In addition, Ret9 and Ret51 regulated distinct gene expression profiles upon ligand stimulations. In this study, a novel Ret serine residue has been identified to be critical for Ret kinase activity and GDNF-induced neurite outgrowth in PC12 cells. These results further demonstrate the functional differences and significance of Ret isoforms in GDNF-induced neurite outgrowth. In the attempt to explore the effects of GFLs and microRNA (miRNA), a novel high performance quantitative real time PCR assay was developed. Using this assay, a number of miRNAs were found to be regulated by GDNF in human glioma cells, which may contribute to the modulation of glioma behaviors by GDNF signaling. The results from this study highlight the functional diversity of GDNF receptor isoforms in regulating neuronal differentiation and glioma migration. The existence of multiple alternatively spliced isoforms of GFRa1, GFRa2 and Ret provides a paradigm shift of how this limited number of ligands and receptors can generate the pleiotropic effects by combinatorial interactions of the various components.