ABBREVIATIONS GDNF glial-cell-line-derived neurotrophic factor PSP persephin ART artemin NTN neurturin GFRα GDNF Family Receptor Alpha GPI glycosyl phosphatidyl inositol CHO Chinese Hamster Ovary DMEM Dulbecco's Minimal Essential Medium SPA scintillation proximity assay SPR surface plasmon resonance RACE rapid amplification of cDNA ends RT-PCR reverse transcription polymerase chain reaction TGFβ transforming growth factor beta by guest on July 18, 2018 http://www.jbc.org/ Downloaded from

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... The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidyl inositol (GPI)-anchored membrane proteins, the GDNF family receptors alpha (GFRα) and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of [ 125 I]-GDNF from GFRα1 is increased in the presence of 50 nM GDNF, an effect that can be explained by the occurrence of negative co-operativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low [ 125 I]-GDNF concentrations suggesting the presence of positive co-operativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pM) and may have an important role in cRET independent signalling. A high affinity site with a K D of 11 pM for [ 125 I]-GDNF is detected only when GFRα1 is co-expressed with cRET at a DNA ratio 1:3. These results suggest an interaction of GFRα1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.