Cloning, mRNA distribution and chromosomal localisation of the gene for glial cell line-derived neurotrophic factor receptor [beta], a homologue to GDNFR-[alpha]
Cloning, mRNA distribution and chromosomal localisation of the gene for glial cell line-derived neurotrophic factor receptor [beta] , a homologue to GDNFR- [alpha]Petro Suvanto1,*, Kirmo Wartiovaara1,2, Maria Lindahl1, Urmas Arumäe1, Maxim Moshnyakov1, Nina Horelli-Kuitunen3, Matti S. Airaksinen1, Aarno Palotie3, Hannu Sariola1,4 and Mart Saarma1
1Institute of Biotechnology and 2Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland, 3Department of Clinical Chemistry and 4HUCH Diagnostics, University Hospital of Helsinki, Helsinki, Finland
Received March 10, 1997;Revised and Accepted May 15, 1997
DDBJ/EMBL/GenBank accession nos AF003825 and U93703
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.
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor [beta] (TGF-[beta]) superfamily, was discovered as a potent survival factor for central dopaminergic neurons (1 ). As a neurotrophic factor, GDNF is expressed in the developing central nervous system but it is also found in many peripheral tissues including embryonic kidney, gastrointestinal tract and skeletal muscle (2 ,3 ). GDNF and the genes responsible for its signal transduction are of great clinical interest due to their potential use in therapy for motoneuron and Parkinson's diseases. In addition, these genes are intensively studied as possible candidate disease genes for congenital or inherited disorders affecting the survival of the neurons in substantia nigra and in the gastrointestinal tract.
Rat GDNFR-[alpha] sequence (6 , GenBank accession number U59486) was used to screen the human EST database and eight human sequences from six different clones (GenBank accession numbers H12981, H05619, R02135, R02249, T03342, W73681, W73633 and Z43761) with high similarity (identity >70% over 200 bp) were identified. Full sequence analysis of three EST clones revealed 3'-terminal consensus sequence named human EST GDNFR-[beta] cDNA (nucleotides 469-1490 in Fig. 1 A). To obtain complete cDNA, human EST GDNFR-[beta] cDNA was used for screening adult rat hippocampus cDNA library. Two identical clones out of one million contained 2002 bp long sequence with 91% identity to probe. Rat cDNA sequence for GDNFR-[beta] (GenBank accession number AF003825) lacked ~60 3'-terminal nucleotides of the open reading frame judged from human EST GDNFR-[beta] and GDNFR-[alpha]. With a forward primer designed from rat GDNFR-[beta] sequence, 5' end of human GDNFR-[beta] was amplified by PCR from human total brain cDNA. The human GDNFR-[beta] cDNA sequence (GenBank accession number U93703) contains a 1395 bp long open reading frame (Fig. 1 A). At amino acid level, human and rat GDNFR-[beta] orthologues were 96% identical. The predicted 47 kDa (unglycosylated) mature protein consists of 464 amino acids that are 48% identical and up to 63% similar to the published sequence of human and rat GDNFR-[alpha] proteins (6 ) (Fig. 1 B). The amino acid sequence of GDNFR-[beta] has a putative signal sequence, three N-glycosylation sites, and a putative GPI-anchor site similar to GDNFR-[alpha]. Completely conserved cysteine residues and strong overall resemblance to GDNFR-[alpha] predict high similarity in the spatial structures of the GDNF-receptors [alpha] and [beta] (Fig. 1 B).
The tissue distribution of human GDNFR-[beta] was studied by northern hybridisation of mRNA extracted from different adult and fetal tissues. The expression of GDNFR-[beta] mRNA was abundant in adult brain, intestine and placenta, as well as in fetal brain, lung and kidney. Two major transcripts of 2.9 and 3.5 kb were visible in all these tissues, and additional transcripts of 7.5 kb in placenta and 1.4 kb in testis (Fig. 2 ) were found.
We have characterised the human and rat cDNA sequences of a new GDNF signal mediating receptor and shown its mRNA expression in multiple adult and fetal tissues that are known to express GDNF and the GDNF receptor tyrosine kinase Ret. Similarly to the earlier characterised GDNF-binding protein, GDNFR-[alpha] (6 ,7 ), GDNFR-[beta] participates in the GDNF-induced autophosphorylation of Ret receptor tyrosine kinase. Unique mRNA expression pattern during embryonic development in several organs including adrenal gland, kidney and gut as well as in nervous system, suggests a functionally independent role for GDNFR-[beta].
Cytokine and growth factor receptors are usually quite complex allowing much redundancy in the choice of ligands and signalling molecules. Of neurotrophic factors, in the neurotrophin (NT) family (22 ,23 ), one of the ligands, NT-3, can activate all three Trk tyrosine kinase receptors, while one of the receptors, TrkB, is a high-affinity receptor for at least two neurotrophins. Additionally, neurotrophins signal through low-affinity neurotrophin receptor p75. In the case of cytokines ciliary neuronotrophic factor (CNTF) and leukemia inhibitory factor (LIF), the picture is even more perplexing (23 ). CNTF and LIF use the same receptor molecules (gp130/LIFR-[beta]), but in addition, CNTF needs a non-signalling partner (CNTFR-[alpha]) to form a complex with gp130, which is a receptor tyrosine kinase also transducing signals of IL-6 and oncostatin M.
GDNFR-[alpha] and GDNFR-[beta] resemble non-signalling components of CNTF/LIF receptors, since they lack apparent signal transducing properties (25 ). Thus, the GDNF induced activation of signalling receptor Ret is mediated by at least two non-signalling receptors. Simultaneous presence of two similarly behaving GDNF-presenting proteins may lower the amount of GDNF needed to activate Ret. At least for spinal motoneurons, which contain mRNAs for all three GDNF receptors, very low concentrations (15 fM) of GDNF are needed for survival (26 ). In accordance, mRNA levels of GDNF in skeletal muscles are quite low (2 ). On the other hand, in the ureter buds of developing kidney, only GDNFR-[alpha] is present with Ret and GDNF mRNA levels being extremely high. The fact that GDNFR-[beta] mRNA is present in some organs (such as adrenal cortex) where GDNF is not available points to the possibility that some other ligand (such as the GDNF homologues neurturin and persefin) may use GDNFR-[beta] in their signal transduction. Likewise, GDNFR-[beta] may also be used in the activation of signalling receptors other than Ret. The relations between GDNF, its homologues and their receptors remain to be determined.
At present, there is no candidate disease assigned to the human locus for the GDNFR-[beta] gene on 8p21-22, but since it probably participates in the signal transduction of GDNF in neurons, the new receptor GDNFR-[beta] is likely to be of great interest in investigations concerning neurodegenerative diseases. In addition, the gene for GDNFR-[beta] is a potent candidate disease gene for congenital disorders that resemble the phenotypes of GDNF or Ret knock-out mice (e.g. Hirschsprung disease, kidney aplasia and dysplasia) and we are screening for mutations in these developmental disorders.
The I.M.A.G.E. EST bacteria clones were obtained from UK MRC Human Genome Mapping Project Resource Centre, Cambridge and were amplified by standard methods. Plasmids were purified with Wizard Miniprep purification kit (Promega) and sequenced with the A.L.F. system (Pharmacia). Two of the EST clones (GenBank accession numbers H12981 and R02249) produced identical, 1032 bp long sequences (Fig. 1 A). The third clone (GenBank accession number W73681) contained shorter, but identical sequence over the same area. EST databank sequences of the same clones were partial and contained numerous minor errors (including short deletions), which prevented the determination of the presence of a proper reading frame.
The adult rat hippocampus [lambda] ZAP cDNA library (Stratagene) was plated, blotted to nylon membrane (Amersham), and hybridised with human EST GDNFR-[beta] probe (Fig. 1 A). Two plaques out of one million hybridized to the probe were replated and purified, pBK-plasmids were excised with helper virus, amplified, purified and sequenced with the A.L.F. system (Pharmacia).
Human brain total RNA was extracted by standard methods and reverse transcribed in a random-primed reaction as described in Superscript II (Life Technologies) protocol. The human GDNFR-[beta] gene was amplified from cDNA under the following PCR conditions: dNTPs in 200 [mu]M concentration and primers (forward) 5'-ATGATCTTGGCAAACGCCTTCTG-3' and (reverse) 5'-TTGCAGTTGTCATTCAGGTTGC-3' in 1 [mu]M concentration, ~5 ng human brain cDNA, 1 U Dynazyme (Finnzymes) Taq polymerase in 50 [mu]l. The 30 cycles after an initial 5 min at 94oC consisted of 30 s at 94oC, 30 s at 57oC and 1 min at 72oC with a final 5 min extension at 72oC. The PCR fragments were cloned into pGEM-T vector (Promega) and four different clones were sequenced. With several primer pairs complete GDNFR-[beta] cDNA sequence was amplified by PCR from the same human brain cDNA. This sequence was identical to the EST-derived sequence. The overlapping inserts of EST and PCR fragments were combined and cloned to get the contig of the full-length human cDNA.
Human full-length GDNFR-[beta] cDNA was cloned into pCDNA3 (Invitrogen) and pBK-CMV (Stratagene) mammalian expression vectors. Rat GDNFR-[beta] cDNA was cloned into the same expression vectors. In one rat construct, 3' end of human GDNFR-[beta] cDNA was added using a unique BclI restriction site, and in another construct an artificial stop codon was inserted instead of the GPI-tail, producing an apparently soluble form of rat GDNFR-[beta].
For northern hybridisation 100 ng of the human EST GDNFR-[beta] insert was labelled with [32P]dCTP (Amersham) by Prime-a-Gene kit (Promega). The specific activity of the final probe was 2 * 107 c.p.m./[mu]g and the hybridisation of Human and Human Fetal Multiple Tissue Northern Blot filters (Clontech) was performed in ExpressHyb solution at 65oC for 2 h. The filters were washed twice for 30 min at 50oC in 2* saline sodium citrate (SSC) + 0.1% SDS and 0.1* SSC + 0.1% SDS and then analysed by phosphoimager (Fuji BAS 1500). As a control, the same filters were hybridised with human [beta]-actin probe (Clontech).
Human peripheral blood lymphocytes were cultured and a cell culture from mouse fetal tissue was established according to standard protocols (26 ) and used as a source of metaphase chromosomes. Both human lymphocytes and mouse monolayer cells were treated with 5-bromodeoxyuridine at early replicating phase to induce a banding pattern (27 ,28 ). The slides were stained with Hoechst 33258 (1 [mu]g/ml) and exposed to UV-light (302 nm) for 30 min. The probes were labelled by a nick translation kit (BRL) with biotin-11-dUTP (Sigma) and the FISH procedure was carried out in 50% formamide, 10% dextran sulphate in 2* SSC as described earlier (29 ,30 ). Repetitive sequences were suppressed with 10-fold excess of Cot-1-DNA (BRL) and after overnight incubation unspecific hybridisation signals were eliminated by washing the slides with 50% formamide/2* SSC, 2* SSC and 0.5* SSC at 45oC. Specific hybridisation signals were visualised using FITC-conjugated avidin (Vector Laboratories) and slides were counterstained with 4'-6'-diamino-2-phenylindole (25 ng/ml). The image analysis for acquisition, display and quantification of hybridisation signals was performed with a PXL camera (Photometrics) attached to a PowerMac 7100/AV workstation. IPLab software controls the camera operation, image acquisition and Ludl wheel (31 ). The probe for human GDNFR-[beta] gene was 1490 bp long cDNA and the hybridisation showed specific double spot signal in 30 out of 100 metaphase chromosomes that were identified based on their G-banding pattern. The hybridisation signal of the 10 kb genomic mouse probe showed specific localisation in 27 out of 30 mouse metaphase chromosomes (32 ).
COS-7 cells (5 * 106 cells per experimental point) were cotransfected by electroporation (Bio-Rad) with cDNAs (5 [mu]g each) of Ret and GDNFR-[alpha], Ret and GDNFR-[beta], GDNFR-[alpha] and GDNFR-[beta], or with cDNA of Ret alone and cultured for 48 h. Cellular phosphatases were inhibited by 1 mM Na3VO4 for 1 h, the cells were treated with 100 ng/ml of GDNF (Promega or PeproTech Ltd) for 30 min and lysed in Tris-balanced saline, pH 7.5, containing 2 mM EDTA, 10% glycerol, 1% NP-40, 1% Triton X-100 and protease inhibitors. Proteins immunoprecipitated by anti-Ret antibodies (Santa Cruz) were analysed by western blotting with anti-phosphotyrosine antibodies PY20 (Transduction Laboratories). In experiments with the secreted form of GDNFR-[beta] lacking a GPI anchor, the cells were not washed before GDNF treatment.
In situ hybridization on E17 rat sections was performed exactly as described previously (2 ,33 ). The antisense cRNA probes for rat GDNFR-[alpha] and rat GDNFR-[beta] covered nucleotides 294-1039 of GenBank sequence U59486 and nucleotides 1231-1394 of GenBank sequence (AF003825), respectively.
We thank Eila Kujamäki for her outstanding excellence in the art of in situ hybridisation. We acknowledge Dr Carlos Ibáñez for rat GDNFR-[alpha] cDNA and Dr Vassilis Pachis for human Ret cDNA. This study was supported by the grants of the Foundation for Pediatric Research, Biocentrum Helsinki, the Sigrid Juselius Foundation and the Academy of Finland.
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*To whom correspondence should be addressed. Tel: +358 9 708 59395; Fax: +358 9 708 59366; Email: petro.suvanto@helsinki.fi
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