title: The first cysteine-rich domain of the receptor GFRalpha1 stabilizes the binding of GDNF.
abstract: The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) 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 125I-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 GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. 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 GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 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 GFRalpha1 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 GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.
authors.affiliation: Institute of Biotechnology, Viikinkaari 9, P.O. Box 56, Viikki Biocenter, University of Helsinki, FIN-00014 Helsinki, Finland.
authors.forename: Heidi
authors.initials: H
authors.lastname: Virtanen
authors.forename: Jianmin
authors.initials: J
authors.lastname: Yang
authors.forename: Maxim M
authors.initials: MM
authors.lastname: Bespalov
authors.forename: Jukka O
authors.initials: JO
authors.lastname: Hiltunen
authors.forename: Veli-Matti
authors.initials: VM
authors.lastname: Leppänen
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
authors.forename: Mart
authors.initials: M
authors.lastname: Saarma
authors.forename: Pia
authors.initials: P
authors.lastname: Runeberg-Roos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000737:chemistry*; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: C495040:Gdnf protein, mouse; C494988:Gfra1 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D003545:Cysteine