Cell Line-derived Neurotrophic Factor Receptor Research Articles

Investigation of the genes for RET and its ligand complex, GDNF/GFRα‐1, in small cell lung carcinoma

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFRα-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13–16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFRα-1, in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines. Genes Chromosomes Cancer 21:326–332, 1998. © 1998 Wiley-Liss, Inc.

Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor that influences the survival and function of several neuronal populations in the central (CNS) and peripheral nervous systems. The actions of GDNF are mediated by a multicomponent receptor complex composed of the tyrosine kinase product of c-ret and the ligand-binding protein GDNF receptor alpha (GDNFR-alpha). In the present study, we used in situ hybridization to localize cells expressing the mRNA for these GDNF receptor subunits in rat CNS. As reported previously, GDNFR-alpha and c-ret mRNA are present in the substantia nigra and ventral tegmental area, regions containing GDNF-responsive dopamine neurons. However, both mRNA were found in motor neurons of spinal cord and brainstem nuclei that innervate skeletal muscle. These areas include alpha motor neurons in the ventral horn of spinal cord and neurons in hypoglossal, facial, trigeminal, and abducens nuclei. In areas rostral to the substantia nigra, c-ret mRNA is not detected, whereas GDNFR-alpha is found in numerous brain structures, including the hippocampus, cortex, medial geniculate, and the medial habenula, the latter area expressing the highest levels of GDNFR-alpha mRNA in brain. These results provide evidence that c-ret and GDNFR-alpha mRNA are expressed in neuronal populations involved in motor function and provides further support for GDNF as a target-derived neurotrophic for these motor neurons. The observation that GDNFR-alpha mRNA is localized in several brain structures that do not contain detectable levels of c-ret mRNA indicates that either GDNFR-alpha utilizes signal transduction molecules other than c-ret in these areas or that other GDNF-like ligands that utilize GDNFR-alpha as a receptor may be present.

Cloning of a novel murine isoform of the glial cell line-derived neurotrophic factor receptor.

We report here the cloning of a novel form of the murine glial cell line-derived neurotrophic factor (GDNF) receptor. Northern blot analyses of various mouse tissues, including whole brain, demonstrated the existence of multiple transcripts of GDNF receptor. Screening of an adult mouse liver cDNA library yielded two isoforms of the receptor. One of the forms (alpha) shows a high degree of homology with other mammalian GDNFR-alpha and the other novel form (beta) is identical to the alpha form except for a deletion of five amino acids. These two forms do not share high sequence homologies with the recently isolated neurturin receptor. Both the alpha and beta forms are expressed in various murine tissues but not in muscle.

The messenger RNAs for both glial cell line-derived neurotrophic factor receptors, c- ret and GDNFRα, are induced in the rat brain in response to kainate-induced excitation

Glial cell line-derived neurotrophic factor (GDNF) has two receptors, receptor-tyrosine kinase c- ret and glycosylphosphatidylinositol-linked cell surface receptor GDNFRα. Kainate-induced seizures, a widely studied model of neuronal plasticity and human epilepsy, have been shown to increase gene expression of several trophic factors, including GDNF, in the rat hippocampus. Here we show that systemic kainate-induced excitation leads to a transient increase of both c- ret and GDNFRα messenger RNAs in the rat brain. Northern analysis demonstrated that, in the hippocampus, the maximal 2.5-fold increase of c- ret and four-fold increase of GDNFRα messenger RNAs was observed after 12 h of kainate injection, in contrast to GDNF messenger RNA, which reaches its maximum in 4–6 h. The blocking of de novo protein synthesis by cycloheximide inhibited the induction of GDNF receptors by kainate, whereas blocking of the N-methyl- d-aspartate-type glutamate receptors by the antagonist dizocilpine maleate did not significantly alter the response. Thus, GDNF receptor messenger RNA increase by kainate depends on protein synthesis, but is not mediated by the N-methyl- d-aspartate receptor. GDNFRα and c- ret show distinct, but partially overlapping, patterns of expression in the brain after kainate treatment. GDNFRα, messenger RNA was prominently induced in the dentate gyrus of the rat hippocampus, less in the habenular and reticular thalamic nuclei and cerebral cortex as revealed by in situ hybridization. C- ret transcripts were induced in the hilus of the hippocampus, several thalamic and amygdala nuclei and in superficial layers of the piriform cortex. These data suggest that GDNF and its receptors may play a local role in neuronal plasticity and in neuronal protection following epileptic insults.

Cloning, mRNA distribution and chromosomal localisation of the gene for glial cell line-derived neurotrophic factor receptor beta, a homologue to GDNFR-alpha.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for central dopaminergic neurons, motor neurons and several other populations of neurons in the central and peripheral nervous system. GDNF and its receptor complex of c-RET tyrosine kinase and a glycosyl-phosphatidylinositol linked protein GDNFR-alpha are of great interest due to their potential use in the therapy of Parkinson's and motoneuron diseases. We have cloned the human and rat cDNA sequences of GDNFR-beta, a new gene encoding for a 464 amino acid long homologue of GDNFR-alpha, and assign the locus of this new gene to human chromosome 8p21-22 and mouse chromosome 14D3-E1. Similarly to GDNFR-alpha, GDNFR-beta mediates GDNF-induced Ret autophosphorylation in transfected cells. By northern hybridisation we show that the transcript level of human GDNFR-beta mRNA is high in the adult brain, intestine and placenta and in fetal brain, lung and kidney. Studied by in situ hybridisation, GDNFR-beta mRNA shows in E17 rat embryo different distribution to that of GDNFR-alpha mRNA, especially, in adrenal gland, kidney and gut. In the developing nervous system, GDNFR-beta mRNA expression is restricted to certain neuronal populations, while GDNFR-alpha mRNA is widely expressed also in non-neuronal cells. The distinct tissue distribution of GDNFR-beta mRNA and its ability to mediate GDNF signal in transfected cells suggest a role in signal transduction of GDNF and, possibly, related neurotrophic factors in vivo.

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs.

Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-alpha), after which the GDNF-GDNFR-alpha associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-alpha mRNA. Ret mRNA is strongly expressed in dopamine neurons and alpha-motor neurons as well as in thalamus, ruber and occluomotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-alpha mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-alpha mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-alpha was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-alpha mRNA did not always match that of Ret mRNA. For instance, GDNFR-alpha mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-alpha might also have functions unrelated to Ret.

Ret expression in the central nervous system

The ret proto-oncogene product (Ret) has been shown to be one of the glial cell line-derived neurotrophic factor (GDNF) receptors in dopaminergic, norepinephric and motor neurons. We immunohistochemically examined the expression of Ret in the human central nervous system (CNS). The distribution of Ret was generally identical to that of myclin as stained using the Klüver-Barrera method. We further investigated the expression of Ret in human fetal brains (19, 29 and 39 weeks gestation) and various brain tumors. The Ret positivity was observed to be associated with the myelin sheath of the cerebral white matter in 29-and 39-week-old fetal brains. Ret is known to be expressed in neural crest-derived cells. We could immunohistochemically confirm the Ret expression in the pheochromocytomas and neuroblastomas of retroperitoneal space. As for the brain tumors, no Ret expression was observed in glioblastomas, oligodendrogliomas, and schwannomas examined, although the glial cells surrounding the tumor and the pre-existing myelin sheath revealed positivity for Ret. In the CNS, Ret expression appears to be closely associated with the myelin sheath; therefore, Ret immunostaining may be useful in ascertaining the demyelinating lesions in the CNS.