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

Abstract

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 fulllength 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.

Lentiviral transfer of an inducible transgene expressing a soluble form of Gas1 causes glioma cell arrest, apoptosis and inhibits tumor growth

“…To obtain a secretable form of Gas1, we generated truncated forms of Gas1, lacking the GPI-link consensus sequence, so these forms of Gas1 will be secreted from the cells. 14,38,39 The autocrine and paracrine effects of Gas1 will significantly increase its therapeutic capacity, by allowing the diffusion and distribution of the therapeutic molecule away from producer cells. To reach this objective we generated lentivirus expressing a truncated form of Gas1 (Arg 315), and determined that infected cells release a soluble form of Gas1 that can induce cell arrest and apoptosis when transferred to independent cultures of C6 cells.…”

“…[7][8][9][10] We also showed that Gas1 exhibits significant structural homology with Glial-cell-line-derived neurotrophic factor (GDNF) receptors (GFRas) and demonstrated that Gas1 inhibits GDNF-mediated intracellular signaling cascades, therefore, shutting down cellular survival pathways. [10][11][12] As the effect of Gas1 interfering with the function of the GFRa-GDNF-Ret complex is due to its structural similarity to GFRas, and it is known that GFRa1 can be released from cells and in this soluble form binds and interacts with GDNF, at the level of the first cysteine-rich domain of the GFRa1, 13,14 we hypothesized that a secreted, soluble Gas1 protein, could exert its inhibitory effect both in the cells that produce soluble Gas1 and in neighbor cells, thus extending its therapeutic capacity to treat tumors. Moreover, there is a report indicating that soluble Gas1, produced in COS7 cells, is functional.…”

“…The lentiviral vector encodes a Gas1 protein truncated at arginine 315, lacking the GPI consensus sequence, thus, this protein will not be anchored to the cell membrane, and will be secreted from the cell. 14,16 Lentiviral vectors effectively infected C6 glioma cells, which produced a protein capable of inducing cell death in in vitro assays. Conditioned media taken from infected cultures caused cell death in independent cultures of glioma cells.…”

RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface

“…The rat GFRa1 construct used in this work (accession number AJ002072) was tagged with the FLAG epitope and has previously been characterized (Virtanen et al, 2005). The human RET constructs (long isoform, accession number NM_000323) were from Marc Billaud (Pelet et al, 1998), and were subcloned into pCR3.1.…”

“…All point mutations were introduced by inverse PCR mutagenesis, and all DNA constructs were sequenced throughout. PC6-3 cells (a subclone of PC12 cells) do not express GFRa1 and have very low levels of endogenous RET (Virtanen et al, 2005). All transfections were carried out using Lipofectamine TM 2000 (Invitrogen, Carlabad, CA, USA) unless otherwise indicated.…”