Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions.
The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), 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 GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. 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 signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.
Cik
M
M
Department of Biochemical Pharmacology and the Department of Biotechnology and High-Throughput Screening, Janssen Research Foundation, B-2340 Beerse, Belgium. mcik@janb.jnj.com
Masure
S
S
Lesage
A S
AS
Van Der Linden
I
I
Van Gompel
P
P
Pangalos
M N
MN
Gordon
R D
RD
Leysen
J E
JE
D003001:Cloning, Molecular; D029721:Drosophila Proteins; D015723:Gene Library; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051101:Neurturin; D020033:Protein Isoforms / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D011869:Radioligand Assay; D020239:Receptor Cross-Talk; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013378:Substantia Nigra / Q000378:metabolism
D029721:Drosophila Proteins; C495039:GDNF protein, human; C494987:GFRA1 protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D007457:Iodine Radioisotopes; C495053:NRTN protein, human; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D051101:Neurturin; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila