Shuqian Jing

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF

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.

GFRalpha-2 and GFRalpha-3 are two new receptors for ligands of the GDNF family.

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

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret.

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprungs disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 fibroblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-deficient RET mutant, associated with Hirschsprungs disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42.

The candidate neuroprotective agent artemin induces autonomic neural dysplasia without preventing peripheral nerve dysfunction.

Artemin (ART) signals through the GFRα—3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfrα—3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.