We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFR␣-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFR␣-1 or GFR␣-2, a novel receptor related to GFR␣-1. Both GFR␣-1 and GFR␣-2 mediate neurturininduced Ret phosphorylation. GDNF can also bind to either GFR␣-1 or GFR␣-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFR␣-1 bind GDNF more efficiently than neurturin, while cells expressing GFR␣-2 bind neurturin preferentially. Crosslinking and Ret activation data also suggest that while there is cross-talk, GFR␣-1 is the primary receptor for GDNF and GFR␣-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFR␣ receptor family. This putative receptor, designated GFR␣-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFR␣-1 and GFR␣-2. Analysis of the tissue distribution of GFR␣-1, GFR␣-2, GFR␣-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFR␣s and Ret are expressed in many of the same tissues, suggesting that GFR␣s mediate the action of GDNF family ligands in vivo.

Glial cell line-derived neurotrophic factor (GDNF) and a related factor, neurturin, promote survival of diverse groups of neurons. Both GDNF and neurturin signal via a two-component receptor complex that consists of a ligand-binding GDNF family receptor (GFRalpha-1 or GFRalpha-2) and the receptor protein tyrosine kinase Ret. Recently, a third receptor related to GFRalpha-1 and GFRalpha-2 has also been isolated and designated GFRalpha-3. Although much is known about the interaction among GDNF family factors, Ret, and the alpha-receptors in vitro, it remains unclear about their interactions in vivo. We show here by in situ hybridization that Ret and the alpha-receptors may be colocalized in the same tissues or expressed separately in projecting and target tissues, respectively, indicating that two distinct modes of interaction between Ret and the alpha-receptors exist in vivo. First, Ret may interact with the alpha-receptors expressed in the same cells (termed interaction "in cis") in many tissues and cell populations that respond to GDNF and/or neurturin, such as the substantia nigra, dorsal root ganglia, spinal cord motoneurons, kidney, and intestine. Second, Ret may interact with the alpha-receptors localized in the target neurons (termed interaction "in trans"). In addition, we present evidence in vitro that GFRalpha-1 mediates Ret activation by GDNF in trans. These observations suggest that there are multiple mechanisms regulating the interaction between Ret and the alpha-receptors that mediates the effects of GDNF family trophic factors on the survival and differentiation of cells and on neuron-target interactions in the nervous system.

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Abstract:Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-i 531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BlAcore data suggested that G-90 and B-1531 recognize different epitopes: POE confirmed that B-1531 binds to NH 2-terminal peptides between amino acids 18 and 37, whereas 0-90 does not; BlAcore data showed that B-i 531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-i 531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by 0-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to downregulate GDNF-induced biological activity. Key Words: Glial cell line-derived neurotrophic factor-Monoclonal antibodies-Motoneurons-Dopaminergic neuronsWestern blot-ELISA.