The first cysteine-rich domain of the receptor GFRα1 stabilizes the binding of GDNF

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRα1 (GDNF family receptor α1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125 I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRα1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRα1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRα1 and RET. Moreover, soluble full-length GFRα1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRα1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRα1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRα1 by stabilizing the interaction between GFRα1 and GDNF.