Human Glial Cell Line-derived Neurotrophic Factor Receptor α4 Is the Receptor for Persephin and Is Predominantly Expressed in Normal and Malignant Thyroid Medullary Cells

Abstract

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) ␣ subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFR␣1, neurturin via GFR␣2, artemin via GFR␣3, whereas the mammalian GFR␣ receptor for persephin (PSPN) is unknown. Here we characterize the human GFR␣4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFR␣4 lack the first Cys-rich domain characteristic of other GFR␣ receptors. Unlabeled PSPN displaces 125 I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFR␣4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFR␣4 may restrict the MEN2 syndrome to these cells.The glial cell line-derived neurotrophic factor (GDNF) 1 family ligands GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN) are structurally related neurotrophic factors that signal through a multicomponent receptor composed of the transmembrane receptor tyrosine kinase RET and high affinity glycosylphosphatidylinositol (GPI)-anchored proteins, the GDNF family ␣ receptors 1-4 (GFR␣1-4, reviewed in Refs. 1 and 2). GFR␣4 was first described from chicken and shown to be the preferential receptor for PSPN (3, 4). Recently, we characterized a mouse GFR␣4 receptor (5). It differs from all other GFR␣ receptors, including chicken GFR␣4, being smaller in size and lacking the first Cys-rich domain. Mouse Gfra4 transcripts are expressed in many embryonic and adult tissues but efficient splicing leading to a functional GPI-linked isoform, as well as putative transmembrane and soluble isoforms, occurs only in thyroid and adrenal medulla and in pituitary intermediate lobe (5). In mouse, Gfra4 and Ret are coexpressed only in the thyroid C-cells and adrenal chromaffin cells. In chicken, Gfra4 mRNA is broadly expressed during embryonic development, including the spinal motoneurons and kidney (4). Chicken GFR␣4 also binds mouse PSPN and confers survival response to PSPN in the presence of Ret (3). However, due to different structures of chicken and mammalian GFR␣4, as well as the lack of information about the existence of chicken GFR␣3, ligand specificity of mammalian GFR␣4 cannot be directly extrapolated from experiments with chicken GFR␣4. PSPN mRNA is expressed ...

The Candidate Neuroprotective Agent Artemin Induces Autonomic Neural Dysplasia without Preventing Peripheral Nerve Dysfunction

“…While the neoplastic effects in thyroid glands in MEN2 have been imputed to GFRα-4 rather than GFRα-3 (Lindahl et al, , 2001, our data showing that overexpression of Art in transgenic mice induced multifocal neuronal expansion in adrenal medulla (Figures 9,11,12) introduces the possibility that excessive ART production provides a means of initiating neoplasia in chromaffin cells. The mature adrenal medulla retains a population of undifferentiated chromaffin cells (Chen-Pan et al, 2002), which possess neurogenic capabilities (reviewed in (Martinez and Mog, 2001)); a reasonable hypothesis is that the neuronal metaplasia in 292 BOLON ET AL.…”

A model for GFRα4 function and a potential modifying role in multiple endocrine neoplasia 2

“…This is consistent with the predicted low level requirements for active RET signaling in maintenance of normal thyroid C-cells (Avantaggiato et al, 1994;Nakamura et al, 1994), which could be achieved by low levels of the high efficiency activating receptor WTa, and higher levels of the low efficiency or inhibitory receptors, WTb and WTc. In MTC tumours, there is almost no isoform WTc but relatively high levels of WTa and WTb (Lindahl et al, 2001) which, in our model, would result in increased relative levels of activating or high efficiency receptor and increased RET signaling. Conversely, the relatively higher levels of the SCc isoform, which we predict is activating, would shift the relative ratio of inhibitory and activating isoforms to better favour RET activation, promoting C-cell proliferation at an early stage of tumorigenesis.…”

“…All GFRa4 isoforms lack the first cysteine-rich domain found in these proteins. The WTa isoform is most similar to other GFRas, containing two cysteine-rich domains, spanning seven alpha helices, and the conserved GPI-linkage (Figure 3), and has been shown to bind both RET and its soluble ligand PSPN and to stimulate RET phosphorylation and survival of neuronal cell types (Enokido et al, 1998;Masure et al, 2000;Lindahl et al, 2001). Compared to WTa, WTb lacks helices 4 and 5 (Figure 3) and has an odd number of cysteine residues that would result in an unpaired residue, potentially available for novel interactions.…”

“…Interestingly, Lindahl et al (2001) have shown that GFRa4 isoforms have tissue-specific differences in relative expression levels. In normal thyroid, isoforms WTb and WTc are relatively highly expressed in comparison to WTa.…”

“…GFRA4 has six coding exons that undergo alternative splicing to generate three variants (a, b, and c) in mouse and human but only two (a, b) in rat (Lindahl et al, 2000(Lindahl et al, , 2001Masure et al, 2000). In humans, introns 2, or 2 and 3, may form part of the GFRA4 coding sequence and exons 3 and 4 can be read in multiple reading frames, while a cryptic splice acceptor site in intron 3 can result in inclusion of an 11 bp sequence (exon 4b) in some transcripts (Figure 1).…”