Browsing by Author "Rautanen, Pauliina Eeva Maria"

Growth Differentiation Factor-15 (GDF15) is a neurotrophic factor associated with anorexia and weight loss. It is elevated in obesity and various diseases. It signals by forming a tripartite complex with the coreceptor Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-like (GFRAL) and the receptor Rearranged during transfection (RET). Targeting this pathway has therapeutic potential for the treatment of obesity and anorexia-cachexia syndrome, but many aspects are still unclear. What is the affinity of binding between these proteins? Does GDF15 induce dose- and time-dependent RET phosphorylation and activate intracellular signaling pathways, and are there differences between GDF15 and GDNF signaling, as the different bend angles of their complexes suggest? Can soluble GDF15-GFRAL mediate the effects of GDF15 outside of the brainstem, and what is the function of the short cytoplasmic domain of GFRAL? Furthermore, how well is the pathway evolutionally conserved between species? Binding affinities were assessed with microscale thermophoresis, whereas RET phosphorylation and intracellular signaling assays were performed utilizing immunoprecipitation and western blotting. GFRAL-RET binding is low-affinity (350 nM ± 223) similarly to GFRα1-RET binding (GDNF family receptor alpha-1), whereas GDF15-RET binding without GFRAL does not occur. GDF15 appears to compete for binding to GFRAL or RET, differing from GDNF mechanisms, but noise in the data may have affected the results. The data provide ideas about the ligand-receptor complex formation. Furthermore, RET phosphorylation by GDF15 is dose- and time-dependent. Firstly, the strongest RET and ERK activation occur at GDF15 concentrations typical of disease states. Secondly, RET activation by GDF15 is rapid and sustained like by GDNF activation, whereas ERK activation by GDF15 is rapid and much more transient than by GDNF. Thirdly, AKT activation by GDF15 is much weaker than by GDNF. The differences may be caused by different conformations of binding surfaces for adaptor proteins being available on RET because the bend angles of the complexes are different. Moreover, soluble GDF15-GFRAL does not activate RET, although soluble GDNF-GFRα1 does. Also, the short cytoplasmic domain of GFRAL is not necessary for activating AKT and ERK pathways, but may be needed to activate RET. Furthermore, GDF15 from cynomolgus monkey, but not rat or mouse, activates RET with human GFRAL, indicating sequence similarity in the active site of GDF15. In conclusion, novel aspects of GDF15 signaling and differences between GDF15 and GDNF signaling were discovered.