Human Glial Cell Line-derived Neurotrophic Factor Receptor alpha 4 Is the Receptor for Persephin and Is Predominantly Expressed in Normal and Malignant Thyroid Medullary Cells*

Maria LindahlDagger §, Dmitry PoteryaevDagger §, Liying YuDagger , Urmas ArumäeDagger , Tõnis TimmuskDagger , Italia Bongarzone, Antonella Aiello||, Marco A. Pierotti, Matti S. AiraksinenDagger , and Mart SaarmaDagger **

From the Dagger  Program in Molecular Neurobiology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, FIN-00014 Helsinki, Finland, the  Division of Experimental Oncology and the || Department of Pathology, Istituto Nazionale Tumori, 20133 Milan, Italy

Received for publication, September 11, 2000, and in revised form, December 12, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha  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 GFRalpha 1, neurturin via GFRalpha 2, artemin via GFRalpha 3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha 4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha 4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces 125I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha 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 GFRalpha 4 may restrict the MEN2 syndrome to these cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  receptors 1-4 (GFRalpha 1-4, reviewed in Refs. 1 and 2). GFRalpha 4 was first described from chicken and shown to be the preferential receptor for PSPN (3, 4). Recently, we characterized a mouse GFRalpha 4 receptor (5). It differs from all other GFRalpha receptors, including chicken GFRalpha 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 GFRalpha 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 GFRalpha 4, as well as the lack of information about the existence of chicken GFRalpha 3, ligand specificity of mammalian GFRalpha 4 cannot be directly extrapolated from experiments with chicken GFRalpha 4.

PSPN mRNA is expressed at low levels in many rat tissues, where two transcripts, an unspliced and a functional spliced form are produced (6, 7). PSPN promotes the survival of embryonic motor neurons in vitro and rescues nigral dopamine neurons following neurotoxic injury in vivo but does not support the survival of any peripheral neurons tested (6). However, unlike GDNF and NRTN, PSPN does not induce neurite outgrowth of rat P8 motor axons in organotypic cultures (8).

Somatic rearrangements caused by chromosomal inversions activate the oncogenic potential of RET in human papillary thyroid carcinomas, whereas germline point mutations are responsible for multiple endocrine neoplasia type 2 (MEN2; reviewed in Refs. 9-11). The malignancies associated with these syndromes arise in several RET-expressing neural crest-derived cell populations. However, RET is expressed at high levels in many other cell types which do not show malignant changes in MEN2 (12). MEN2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytomas and parathyroid hyperplasia, whereas MEN2B is characterized by MTC associated with pheochromocytomas, enteric ganglioneuromas, skeletal abnormalities, and mucosal neuromas. MTC is the only phenotype in familial medullary thyroid carcinoma. In MEN2A, cysteine substitutions in the extracellular domain of RET cause aberrant homodimerization and ligand-independent constitutive activation of RET (13, 14), which alone or together with unknown factors cause the malignant proliferation of cells. Familial medullary thyroid carcinoma mutations are more evenly distributed among the extracellular cysteines but are also found in the intracellular part of RET. Most cases of MEN2B contain a specific mutation in the tyrosine kinase domain, which is also frequently found in sporadic MTC. This leads to constitutive activation of RET with altered substrate specificity in downstream signaling pathway (13, 14). However, in MEN2B-RET transfected cells, ligand binding has been reported to increase the intensity of RET signaling (15, 16). Transgenic mouse models of these mutations have shown to cause some of the malignant phenotypes found in human MEN2 syndromes (17-19). The tissue-specific tumorigenesis and phenotypic variability within a MEN2 family and between individuals carrying the same mutation in RET, suggests that further genetic events or modifier genes are required to induce the tumor phenotype.

In this paper, we have characterized the human GFRalpha 4 receptor and show that it is a functional co-receptor for PSPN, which mediates RET activation. Selective coexpression of GFRA4 and RET in normal and malignant C-cells suggests that GFRA4 is a candidate modifier gene in MTC.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RNA Isolation-- RNA from different human tissues was purchased from CLONTECH (Human Total RNA Panels I-V). Total RNA from primary MTC tumors was isolated using UltraspecTM-II RNA isolation system (Biotecx Laboratories, Inc.). Total RNA from the TT cell line (ATCC number CRL1803) was isolated with Trizol reagent (Life Technologies). RNA from human fetal thyroid and adrenal tissue (17- and 18-week-old, respectively) was isolated from autopsy samples (Hospital for Children and Adolescents, University of Helsinki, Finland), by RNAwizTM isolation reagent (Ambion). Total RNA from human blood cells was isolated using the QIAampTM RNA blood mini kit (Qiagen) according to the manufacturer's instructions. The RET mutation of five MTC patients had been analyzed: one from a MEN2A family member contained the germline C634R mutation, one patient was positive for the M918T but the germline situation was unknown, and three were sporadic MTCs of which one was positive for the M918T mutation.

RACE Cloning and RT-PCR-- Human GFRA4 cDNAs were identified from a human thyroid cDNA (CLONTECH Marathon Ready cDNA) by the 3'- and 5'-RACE method using the GC-rich PCR kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Reverse transcription reactions were performed using Superscript II (Life Technologies). PCR systems were GC-rich PCR kit (Roche Molecular Biochemicals) for GFRA4 and PSPN, Expand Long Template PCR system (Roche Molecular Biochemicals) for GFRA1, GFRA2, GFRA3, and RET, and Dynazyme II (Finnzymes) for PPIA (alias cyclophilin). PCR was run 40 cycles for GFRA4, 35 cycles for PSPN, GFRA1, and GFRA2, and 30 cycles for GFRA3, RET, and PPIA using annealing temperatures of 55-60 °C. Full-length human GFRA4 cDNA was obtained using Expand High Fidelity system PCR enzyme (Roche Molecular Biochemicals) together with GC-rich PCR system buffers (Roche Molecular Biochemicals). PCR products were cloned into mammalian expression vector pCR3.1 (Invitrogen).

The antisense primers used in 5'-RACE were the mouse-specific Gfra4 primer 5'-CACGTTGTCCAGGTAGTTGG-3' and a second human-specific GFRA4 nested primer 5'-GCACTGCGCCACATACTCGGA-3'. The 3' region of GFRA4 was cloned in two steps, first using a GFRA4-specific sense primer P2 (see Fig. 1A) 5'-GCTCCGAGTATGTGGCGCAGT-3' and a nested primer 5'-GCTCACCCACGCACTGCTCTTCTG-3'. The 3'-region containing the stop codon and 3'-untranslated region sequence was cloned using a sense primer 5'-CCTAACTACGTGGACAACGTGAGC-3' and a second nested primer 5'-ATGGTGCCATTCAGGCCTTTGCCAG-3' or 5'-GCAGGTGTCCTCCACAGGCAG-3'. RT-PCR of genomic human DNA was used to locate the intronic nucleotide sequences between exons 4 and 5, and between exons 5 and 6, using a sense primer 5'-CCTAACTACGTGGACAACGTGAGC-3' and an antisense primer 5'-GAAGTATGGAGAGCAGGGAGCGTC-3'. Full-length human GFRA4 cDNAs were obtained using a sense primer P1 (see Fig. 1A), 5'-CCACCATGGTCCGCTGCCTGG-3' and an antisense primer P3, 5'-GAGGTCGCTGTCCTAATCAGAG-3'.

Primers used in RT-PCR for human, mouse, and rat PSPN were as described in Ref. 6. Primers used in RT-PCR for human GFRA1 flanked nucleotides 491-870 of GenBankTM sequence AF042080, for GFRA2 flanked nucleotides 148-427 of GenBankTM sequence U93703, for GFRA3 flanked nucleotides 574-1203 of GenBankTM sequence NM001496, and for RET flanked nucleotides 833-1114 of GenBankTM sequence X12949. The amounts of total RNA in samples were normalized by amplification of a PPIA fragment.

125I-Labeled PSPN Binding-- Murine neuroblastoma Neuro-2a cells were transfected (FuGene6, Roche Molecular Biochemicals) with full-length GFRA4a cDNA in pCR3.1 vector (Invitrogen), and bulk selected with 400 µg/ml G418. PSPN was enzymatically iodinated by lactoperoxidase, to a specific activity of 100,000 cpm/ng. Binding assays were performed essentially as described (20) with 0.9 nM 125I-PSPN in binding buffer (Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 15 mM Hepes, pH 7.5) for 4 h on ice, either in the presence or absence of different concentrations of unlabeled human PSPN (PeproTech EC Ltd.), rat GDNF (Cephalon, Inc.), human NRTN (PeproTech EC Ltd.), or human ARTN (a gift from Drs. J. Milbrandt and E. M. Johnson, Jr.). The amount of 125I-PSPN bound to mock-transfected cells was at the background level. Kd was calculated using Cheng-Prusoff equation (21).

Chemical Cross-linking and Immunoprecipitation-- Chinese hamster ovary cells were transfected (FuGene 6) with full-length human GFRA4a cDNA, grown for 2 days, washed and incubated with 0.9 nM 125I-PSPN in binding buffer (see above) on ice for 4 h. After washing the cells were incubated with 1 mM bis-suberate (Pierce) in phosphate-buffered saline at room temperature for 25 min. Following washes, some samples were treated with 0.5 unit/ml phosphoinositide-phospholipase C (Sigma) at +37 °C for 30 min. Cells were lysed in Laemmli buffer containing beta -mercaptoethanol.

The cross-linking reaction for human GFRA4a and mock transfected (pcDNA3; Invitrogen) Neuro-2a cells was done as described above but the cells were lysed in Nonidet P-40 lysis buffer (1 × Tris-buffered saline, 2 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, CompleteTM protease inhibitor mixture, Roche Molecular Biochemicals), clarified by centrifugation at 14,000 rpm for 20 min, immunoprecipitated with 1 µg/ml Ret antibodies (Santa Cruz Biotechnology), and collected with Protein A-Sepharose. The immunocomplexes were washed with Nonidet P-40 buffer, and separated by 7.5% SDS-polyacrylamide gel electrophoresis.

Ret Phosphorylation Assay-- Semiconfluent human GFRA4a-, rat Gfra1-, or vector-expressing Neuro-2a cells were maintained with 0.5% serum for 24 h and then in serum-free medium for 4 h prior to stimulation. After stimulation with PSPN (0.01-100 ng/ml) or GDNF (100 ng/ml), cells were lysed and precipitated with agarose-conjugated anti-phosphotyrosine antibodies (4G10, Upstate Biotechnology) overnight at +4 °C on ice. The membranes were probed with 60 ng/ml anti-Ret antibodies (Santa Cruz Biotechnology).

Neuronal Microinjections-- Superior cervical ganglion (SCG) neurons from postnatal day 1-2 FVB or NMRI strain mice were grown for 5-6 days on polyornithine/laminin-coated dishes with NGF (30 ng/ml). 50:50 ng/µl cDNA mixtures of short isoform of human RET (gift from Dr. M. Billaud) and either mouse Gfra4a1 (in pcDNA3, Invitrogen) or human GFRA4a (in pCR3.1, not shown) or empty pcDNA3 vector were pressure-microinjected into nuclei. To find successfully injected neurons later, a plasmid encoding enhanced green fluorescent protein (10 ng/µl) was included in every injection mixture. Neurons were grown overnight with NGF and thereafter in NGF-free medium containing blocking anti-NGF antibodies (Roche Molecular Biochemicals) and PSPN, GDNF, NRTN, or ARTN, all at 100 ng/ml. Number of living fluorescent neurons or uninjected control neurons was then counted (initial neurons). 30-80 initial neurons were successfully injected for every treatment group. Healthy fluorescent neurons with intact nuclei and phase-bright cytoplasm were counted 70-75 h later and expressed as percentage of initial neurons. Experiments were repeated on independent cultures: n = 7 for PSPN, n = 4 for GDNF and NRTN, n = 3 for ARTN. In each repeat, all treatment groups (plasmid combinations and controls) for a given factor, shown as a separate group of bars on Fig. 4, were assayed collectively in the same experiment. Experiments with GFRalpha 4 alone (with and without PSPN) were performed separately (n = 4) and combined with other PSPN data. Significance of the differences between means was estimated by one-way ANOVA followed by Tuckey's post-hoc test at the significance level of alpha  = 0.05.

In Situ Hybridization-- In situ hybridizations for RET, GFRA1, GFRA2, GFRA3, and GFRA4 were performed as described (22) on cryosections of the same thyroid tumor samples analyzed by RT-PCR. A 206-bp cDNA fragment of the 5'-end of human GFRA4 was used as template for sense and antisense RNA probes. PCR fragments of GFRA1, GFRA2, GFRA3, and RET generated with the same primers as in RT-PCR were cloned and sequenced and used as templates for antisense and sense RNA probes. Control sections hybridized with sense probe did not show labeling above background (not shown). Dark-field and corresponding bright-field images of Nissl counterstained sections were digitized and processed using Adobe PhotoShop software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of Human GFRA4-- Sequence information from the mouse Gfra4 cDNA and gene (5) was used to identify human GFRA4 cDNA and genomic clones. First, the genomic region covering putative exon 2 to exon 4 was cloned by PCR from human genomic DNA. Next, the structure of GFRA4 cDNAs and gene were characterized by 5'- and 3'-RACE of adult human thyroid cDNA and PCR of genomic DNA using primers corresponding to different regions of the putative exons of human GFRA4 (see "Materials and Methods"). The sequence of human GFRA4 exon 1 encoding the signal sequence showed high homology to the mouse Gfra4 exon 1a (5). cDNAs encoding proteins with an alternative signal sequence homologous to the 1b signal sequence found in mouse, or putative transmembrane and soluble isoforms found in mouse (5), were not identified from human thyroid. Instead, three different alternatively spliced GFRA4 cDNAs were identified (GFRA4a, GFRA4b, and GFRA4c, Fig. 1B): (i) GFRA4a (810 bp) corresponds to the mouse Gfra4 transcript a1 (5). The predicted protein (GFRalpha 4a, 290 amino acids, Fig. 1C) contains a putative N-terminal hydrophobic signal, one N-linked glycosylation site (NVSA) at position 178, and a hydrophobic stretch of amino acids in the C terminus, preceded by a hydrophilic linker region, consistent with a GPI-anchor signal sequence (23). The amino acid identity between mouse and human GFRalpha 4a is 76%, whereas the identity between human and chicken GFRalpha 4 is 54% (covering amino acids 143-340 in chicken GFRalpha 4).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Structure of the human GFRA4 gene compared with mouse Gfra4 and different splice forms of human GFRA4 mRNA. A, structure of the human GFRA4 gene (top) compared with mouse Gfra4 gene (bottom). Both contain at least 6 exons but no alternative exon 1b has been found in human. Also the putative transmembrane form encoded by the alternatively spliced exon 6 found in mouse is absent in human. Primers P1-P3, used in RT-PCR in Figs. 2 and 5 are marked by arrowheads. B, splicing of the GFRA4 gene in human thyroid gland. Two GPI-linked isoforms (GFRA4a and GFRA4b), and one putative soluble isoform (GFRA4c) are produced by alternative splicing. In transcript encoding for GFRalpha 4a all the introns are spliced, whereas GFRalpha 4b contains the intron between exons 2 and 3. Transcript encoding GFRalpha 4c contain introns between exons 2 and 3 and between exons 3 and 4. Asterisk marks the stop codon. C, proposed schematic domain structure of human GFRalpha 4 isoforms.

The other identified alternative human GFRA4 transcripts are: (ii) GFRA4b (900 bp), in which the small intron (79 bp) separating exons 2 and 3 (Fig. 1A) is included in the transcript, and the 3'-splice site of exon 4 is located 11 bp upstream of the respective splice site used in the GFRA4a transcript. The small intron between exons 2 and 3 is also inefficiently spliced in the majority of mouse tissues. Inclusion of this intron in mouse Gfra4 transcripts with exon 1a or exon 1b leads to a putative soluble protein isoform (5). In contrast, translation of the transcript GFRA4b in human would lead to a GPI-linked protein of 299 amino acids. In this protein isoform, the N- and C-terminal ends are identical to the respective regions of GFRA4a, but the middle region consists of a stretch of 66 amino acids translated in different frame from intron 2 and exon 3. This sequence is not homologous to any protein in public data bases. (iii) GFRA4c (867 bp), in which the introns between exons 2 and 3, and between exons 3 and 4 are included in the transcript. These introns would lead to a frameshift with a stop codon located inside exon 5 and production of a putative soluble isoform of 236 amino acids. It is interesting to note that the sequences of the 3'-splice sites of both mouse and human exon 3 and exon 4 contain a short polypyrimidine tract interrupted with purines, which is characteristic to alternatively spliced exons (24).

Expression of GFRA4, PSPN, and RET in Different Human Tissues-- Expression of GFRA4, PSPN, and RET was analyzed by RT-PCR in 27 different adult and 4 fetal tissues (Fig. 2 and not shown). Using primers P2 and P3, expression of GFRA4 was detected at high levels in the adult thyroid gland (Fig. 2) and, at lower levels, in the fetal adrenal and fetal thyroid gland (not shown), whereas all other tissues analyzed did not express detectable levels of GFRA4. Similar results were obtained with other combinations of primers P1, P2, and P3 (data not shown). On Fig. 2, the smallest fragment of 699 bp corresponds to the GFRA4a cDNA where all introns are spliced out, whereas the 789-bp fragment corresponds to the intron 2-containing GFRA4b transcript. The 863-bp PCR fragment corresponds to the cDNA containing both introns 2 and 3 encoding the putative soluble isoform (GFRA4c, Fig. 1B). The unspliced transcript (560 bp) of PSPN was expressed in all human tissues examined using primers detecting the full-length PSPN transcript (Fig. 2, second row). No transcripts were observed when RNA was used as a template for PCR, which shows that the cDNA samples did not contain chromosomal DNA. Low levels of spliced PSPN transcripts (476 bp) encoding the functional protein were present in adult human adrenal gland, cerebellum, spinal cord, and testis, and were not detected in the thyroid gland. The nucleotide sequences for the spliced and unspliced PSPN transcripts were verified by sequencing. RET mRNA was expressed at variable levels in most tissues examined (Fig. 2, third row).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 2.   RT-PCR analysis of GFRA4, PSPN, and RET transcripts in various human tissues. Top row, GFRA4 expression is detected only in thyroid gland using primers P2 and P3 (see Fig. 1A). All the introns in the transcript corresponding to 699 bp are spliced. The 789-bp transcript contains a small intron between exons 2 and 3 and corresponds to the GFRalpha 4b form. The 863-bp fragment contains introns between exons 2 and 3 and between exons 3 and 4, and corresponds to the soluble GFRalpha 4c form. Second row, an unspliced transcript of the human PSPN is expressed in all tissues (560 bp), whereas the spliced transcript (476 bp), which corresponds to the functional PSPN, is weakly expressed in cerebellum, adrenal gland, and testis and barely detectable in spinal cord. To exclude DNA contamination in RT-PCR reactions, controls containing RNA were also subjected to PCR (not shown). Third row, RET expression (282 bp) is detectable in almost all human tissues examined but the expression levels are strongest in adult brain, cerebellum, spinal cord, salivary gland, adrenal gland, and prostate. Bottom row, control RT-PCR with PPIA transcript showing equal loading of cDNA. In all PCR experiments, negative water controls were included (not shown).

PSPN, but not Other GDNF Family Ligands Binds and Activates Human GFRalpha 4-Ret Receptor Complex-- To study the binding of human PSPN to human GFRalpha 4, we used GFRA4a-transfected mouse neuroblastoma Neuro-2a cells (hGFRalpha 4/Neuro-2a), which endogenously express Ret. These cells strongly bound 125I-PSPN, while mock-transfected Neuro-2a cells did not (Fig. 3C). Low concentrations (~1 nM) of unlabeled PSPN effectively displaced 125I-PSPN from the GFRA4-expressing cells, whereas GDNF (up to 300 nM), NRTN (up to 200 nM), and ARTN (up to 200 nM) were ineffective (Fig. 3A). Thus, in the presence of Ret, human GFRalpha 4 binds specifically PSPN, but not the other GDNF family ligands. PSPN binds to human GFRalpha 4 with a dissociation constant (Kd) of ~100 pM (Fig. 3A).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Binding and chemical cross-linking of 125I-PSPN to human GFRalpha 4 and Ret. A, displacement binding of 125I-PSPN by PSPN, NRTN, GDNF, and ARTN from human GFRalpha 4a-expressing Neuro-2a cells, shown as percentage of 125I-PSPN bound in the absence of a cold ligand (100%). One representative experiment out of four is shown. B, chemical cross-linking of 125I-PSPN to vector- (lanes 1 and 2) or hGFRA4a transfected (lanes 3-6) Chinese hamster ovary (CHO) cells, in the presence (lanes 2, 3, and 5) or absence (lanes 1, 4, and 6) of unlabeled PSPN, treated (lanes 3 and 4) or not treated (lanes 1, 2, 5, and 6) with phosphoinositide-phospholipase C. Bands are designated as follows: arrowhead, ~46 kDa; arrow, ~62 kDa; asterisk, ~92 kDa. C, proteins cross-linked to 125I-PSPN in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of unlabeled PSPN in vector- (lanes 1 and 2) or GFRA4a- (lanes 3 and 4) transfected Neuro-2a cells. Bands are designated as in B. D, immunoprecipitation of Ret from vector- (lanes 1 and 3) or GFRA4a-transfected (lanes 2 and 4) Neuro-2a cells after chemical cross-linking of 125I-PSPN, separated in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of beta -mercaptoethanol (beta -Me). Bands are designated as follows: arrowhead, ~46 kDa; arrow, ~190 kDa; diamond, ~220 kDa; asterisk, ~400 kDa. E, phosphorylation of Ret in mock- (lanes 1 and 2), GFRA4a- (lanes 3-5) or Gfra1-transfected Neuro-2a cells upon stimulation with 100 ng/ml PSPN (lanes 2, 4, and 5) or 100 ng/ml GDNF (lane 7). WB, Western blot; IP, immunoprecipitation. F, dose-dependent phosphorylation of Ret with PSPN in hGFRA4a-transfected Neuro-2a cells.

Next we studied the binding of PSPN to GFRA4a-transfected Chinese hamster ovary cells, which do not express Ret. Treatment of the cells with 125I-PSPN, followed by chemical cross-linking and analysis by SDS-polyacrylamide gel electrophoresis, resulted in a major band of ~46 kDa and minor bands of ~62 and 92 kDa (Fig. 3B). The ~46-kDa band corresponds a PSPN monomer (~16 kDa) cross-linked to monomeric GFRalpha 4 (30 kDa). The ~62- and 92-kDa bands most probably correspond to PSPN dimer cross-linked to GFRalpha 4 monomer and dimer, respectively. Additional 16- and 32-kDa bands represent PSPN monomer and dimer, respectively (Fig. 3B, lane 6). No specific bands were detected from mock-transfected cells (Fig. 3B, lanes 1 and 2). The amount of cell-bound complexes was greatly reduced by adding unlabeled PSPN or by removal of the GPI-anchored proteins by phosphoinositide-specific phospholipase C after cross-linking (Fig. 3B, lanes 4 and 5). Combination of these two treatments further reduced the yield of cross-linked products to undetectable levels (Fig. 3B, lane 3). Thus, the human GFRalpha 4 protein, encoded by the GFRA4a transcript indeed contains a GPI anchor. Cross-linked complexes of the same sizes were also identified from hGFRalpha 4/Neuro-2a cells (Fig. 3C). In addition, a minor band of about 200 kDa was observed (Fig. 3C) that could be a complex of PSPN-GFRalpha 4 with Ret.

To study the interaction of PSPN with Ret, proteins cross-linked to 125I-PSPN in hGFRalpha 4/Neuro-2a cells were precipitated with Ret antibodies (Fig. 3D, lanes 2 and 4). The major cross-linked complexes of ~190 and 220 kDa, as well as minor complexes of about 62 and 92 kDa (same size as bands in Fig. 3C), and also of ~400 kDa were obtained under reducing conditions (Fig. 3D, lane 2). Under nonreducing conditions, the ~400-kDa band was greatly intensified, which indicates the presence of S-S-bound complexes. No cross-linked cell-bound products were detected using parental Neuro-2a cells (Fig. 3D, lanes 1 and 3). The bands of about 190 and 220 kDa correspond to the complexes of PSPN-Ret (186 kDa) and PSPN-GFRalpha 4-Ret (216 kDa), respectively. The components of the complex of about 400 kDa could be a dimer of the 186-kDa PSPN-Ret complex.

To determine whether PSPN binding to human GFRalpha 4 mediates Ret autophosphorylation, we used hGFRalpha 4/Neuro-2a cells treated with PSPN. Gfra1 expressing Neuro-2a cells treated with GDNF served as a positive control. PSPN induced Ret tyrosine autophosphorylation in hGFRalpha 4/Neuro-2a cells, which showed a phosphorylated band of 170 kDa, corresponding to the active form of Ret (Fig. 3E). No phosphorylation of Ret was observed in vector-transfected Neuro-2a cells (Fig. 3E, mock). Stimulation of Ret phosphorylation was dose-dependent starting at 0.1 ng/ml PSPN (Fig. 3F).

PSPN Specifically Promotes Survival of Neurons Ectopically Expressing GFRalpha 4 and RET-- We further studied whether binding of PSPN to mammalian GFRalpha 4-Ret complex triggers functional cellular programs, as described for chick GFRalpha 4 (3). We expressed the GPI-linked mouse Gfra4 or human GFRA4a together with human RET in neonatal mouse SCG neurons and maintained the neurons further with PSPN for 3 days. SCG neurons are trophically dependent on NGF but not on PSPN (3, 5, 6, Fig. 4). As verified by RT-PCR, neonatal mouse SCG expressed transcripts for Ret but not for Gfra4 (data not shown). Significant portion of neurons overexpressing mouse Gfra4 and RET was maintained by PSPN (60 ± 5, 1% versus 16 ± 1, 2% without PSPN, p < 0.001), whereas omission of Gfra4 abolished this trophic effect (Fig. 4). Similar results were obtained with human GFRA4a (not shown). Moreover, introduction of Gfra4 alone was already sufficient to confer PSPN dependence to the SCG neurons (Fig. 4), obviously via endogenous Ret, whereas without PSPN, the survival rate of Gfra4-injected neurons did not differ from that of uninjected or vector-injected neurons (Fig. 4). GDNF and NRTN also maintained part of neurons overexpressing RET and Gfra4, but these effects were not reduced by omission of Gfra4 (Fig. 4). Moreover, both factors maintained also uninjected neurons similarly to receptor-injected neurons (Fig. 4). ARTN promoted survival of neither GFRalpha 4/RET injected nor uninjected mouse SCG neurons (Fig. 4). Thus, GFRalpha 4-RET complex requires PSPN, but not other GDNF family members, to activate a survival-promoting program in SCG neurons.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   PSPN specifically promotes survival of RET/Gfra4-injected SCG neurons. Neonatal mouse SCG neurons were grown 5 days with NGF, injected with indicated plasmid mixtures (all 50:50 ng/µl), grown overnight with NGF, then NGF was changed for indicated factors (all 100 ng/ml). Numbers of viable neurons after a 3-day treatment period are expressed relative to the number of initial neurons. Mean ± S.E of three to seven independent repeats for each factor is shown. *, p < 0.05; ***, p < 0.001.

Selective Expression of GFRA4 and RET mRNAs in Medullary but not Other Primary Thyroid Tumors-- Co-localization of Gfra4 and Ret in developing and mature mouse thyroid C-cells (5), and their coexpression in adult human thyroid tissue (Fig. 2), prompted us to study the expression of GFRA4, RET, and PSPN mRNAs in human primary thyroid tumors. RT-PCR analysis of GFRA4 expression, using primers generating full-length transcript, showed that similar levels of GFRA4 mRNAs encoding the two GPI-linked isoforms and lower levels of transcripts encoding the soluble isoform, are expressed in all eight MTC samples analyzed (Fig. 5, first row). GFRA4 mRNA was not detected in any of the follicular thyroid adenomas (FTA), follicular thyroid carcinomas (FTC), or papillary thyroid carcinomas (PTC) analyzed. Cellular localization of GFRA4 mRNA was studied by in situ hybridization in the same thyroid tumor samples (Fig. 6, A and B). GFRA4 was highly and evenly expressed by virtually all the malignant C-cells in the MTC samples analyzed, but not in the accompanying connective tissue and blood vessels. No expression of GFRA4 was detected in the adjacent apparently normal follicle cells found in some samples. Other thyroid tumors, including FTA, FTC, and PTC did not express GFRA4. None of the thyroid tumors expressed detectable levels of spliced PSPN mRNA, whereas the unspliced form of PSPN transcript was present in all tumor samples analyzed (Fig. 5, second row). High levels of RET expression were seen in all MTC tumors (Fig. 5, third row, and Fig. 6).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5.   Semiquantitative RT-PCR analysis of GFRA4, PSPN, and RET in different human thyroid tumors. Top row, GFRA4 mRNA is selectively expressed in MTC. No GFRA4 transcripts are detectable in FTA, FTC, or PTC. Each lane represents tumor cDNA from different patient. Normal thyroid gland, including medullary tissue (NT) also expresses GFRA4. Two major transcripts of 831 and 910 bp seen in MTC and TT cells correspond to GFRalpha 4a and GFRalpha 4b, respectively (primers P1 and P3, see Fig. 1 A). The minor transcript of 999 bp corresponds to the soluble GFRalpha 4c. Second row, no spliced form of the PSPN transcript is detectable using primers covering the initiation and the stop codons. Third row, RET mRNA is expressed in all MTC samples. Bottom row, control RT-PCR with PPIA transcript showing equal loading of cDNA. All experiments contained negative water controls (not shown).


View larger version (132K):
[in this window]
[in a new window]
 
Fig. 6.   Localization of GFRA1, GFRA2, GFRA3, GFRA4, and RET mRNAs in thyroid tumors by in situ hybridization. A, typical representative examples of autoradiography films showing GFRA1-GFRA4 and RET cRNA hybridization to adjacent frozen sections of different thyroid tumors. Of the four types of tumors represented, only the MTC sections were strongly stained for GFRA4 and RET expression. GFRA1, GFRA2, and GFRA3 mRNA levels were very low or undetectable in all samples including MTC. B, GFRA4 and RET probes were hybridized to frozen sections representing FTA, FTC, PTC, and MTC samples and sections were counterstained by Nissl substance. GFRA4 and RET is highly expressed in all malignant cells of the MTC sample but not in the surrounding connective tissue. Bar in A = 0.5 cm, in B = 100 µm.

Tumor samples were further analyzed for GFRA1, GFRA2, and GFRA3 mRNA expression, by RT-PCR and in situ hybridization (Fig. 6A and Table I). In some tumor samples, low levels of GFRA1, GFRA2, and GFRA3 mRNAs were present in subsets of the tumor cells. To study if the GFRA4 gene is mutated in the MTC tumors, full-length GFRA4 cDNA clones from eight MTC samples, the TT cell line, and normal thyroid were sequenced. Except for a few polymorphisms in the coding region, which do not change the amino acid composition of GFRalpha 4, no mutations were found in the MTC samples or in the TT cell line. Thus, the coding region of GFRA4 gene is not mutated in MTC tumors.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Expression of human GFRA1, GFRA2, GFRA3, GFRA4, and RET in thyroid tumors and normal thyroid
mRNA expression is graded from; -, no labeling above background; to +++, highest detected expression level based on visual inspection. The numbers below the tissue gradings denote the number of samples positive/total number of samples analyzed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Structure and Expression of Human GFRalpha 4-- In this study, we describe the cloning of human GFRalpha 4 cDNAs encoding three different isoforms of human GFRalpha 4, two GPI-linked and one putative soluble isoform, from adult human thyroid gland. We have earlier described the cloning and expression of various mouse GFRalpha 4 cDNAs encoding different protein isoforms (5). As in mouse, all human GFRalpha 4 isoforms lack the first Cys-rich domain (D1) common for all known GFRalpha receptors, including chicken GFRalpha 4 (4).

Of 27 different adult and 4 fetal tissues studied, GFRA4 transcripts were mainly detected in the adult thyroid gland, but also at low levels in the fetal adrenal and thyroid glands. However, although high levels of Gfra4 are present in the intermediate lobe of the mouse pituitary (5), we did not find GFRA4 mRNA in the adult human pituitary, possibly due to the rudimentary nature of the pituitary intermediate lobe in human. Low levels of GFRA4 transcripts were also detected in fetal but not in the adult human adrenal gland, whereas high Gfra4 levels are found in corresponding mouse tissue (5). This apparently predominant expression of human GFRA4 in thyroid and adrenal glands suggests a more tissue-specific transcriptional regulation and possibly a more restricted role for GFRalpha 4 in human, than in chicken and rodents, where Gfra4 transcripts are expressed in many tissues (4, 5). Consistent with this, no human ESTs for GFRA4 were present in public data bases in contrast to several mouse ESTs for Gfra4 (5).

In agreement with previous results (12, 25, 26), RET mRNA was expressed at variable levels in many adult human tissues, including the thyroid and adrenal glands. The functional spliced PSPN mRNA has previously not been identified in human (6). In this study, we found the unspliced transcript of PSPN in all human tissues examined. However, we could also detect low levels of the spliced PSPN mRNA in human adrenal gland, cerebellum, spinal cord, and testis. In contrast, similar levels of both PSPN transcripts are present in most tissues in rat (6, 7). Although the spliced transcript encoding functional PSPN is expressed in the rat thyroid (not shown), we could not detect its expression in either adult human (Fig. 2) or postnatal mouse (not shown) thyroid gland. Adrenal gland was the only tissue where the spliced PSPN transcript was detected in all three species analyzed (mouse, rat and human). Thus, the source of ligand for GFRalpha 4 in human thyroid remains elusive. It is possible that functional PSPN is expressed in the thyroid region at developmental stages not analyzed here due to the lack of commercially available RNAs.

After completion of this study, a Gfra4 cDNA encoding a different GFRalpha 4 isoform was cloned from rat brain and reported as a GPI-linked receptor for PSPN (27). However, the signal sequence encoded by the rat Gfra4 cDNA in that report (27) is very weak and differs from the GFRalpha 4 signal sequence we have identified by 5'-RACE cloning from rat thyroid (MACCLESALLLLLLLLLLGSASS).2 The rat GFRalpha 4 signal sequence identified by us is strong and shows high homology to the mouse (76%) and human (67%) signal sequences encoded by exon 1a in mouse and by exon 1 in human, suggesting that this rat GFRalpha 4 signal sequence is also functional. The putative rat GFRalpha 4 signal sequence identified by Masure et al. (27) shows 59% amino acid identity with the putative signal sequence encoded by the alternatively spliced exon 1b of mouse Gfra4 (5). Our results suggest that both mouse and rat Gfra4 genes have two 5'-exons encoding alternative N termini of the GFRalpha 4 protein. In human, however, GFRA4 cDNAs encoding alternative signal sequences were not identified.

Masure et al. (27) identified only the exon IB containing rat Gfra4 transcript and showed that high levels of Gfra4 mRNA are present in the brain using cRNA probe specific to the region that is present in all Gfra4 alternatively spliced transcripts. They did not study if the Gfra4 transcripts expressed in brain encode functional protein isoforms with signal sequence. We have shown that in mouse the transcripts containing exon Ia or exon Ib are expressed at extremely low levels in the nervous system. Furthermore, we have shown that although high levels of Gfra4 mRNA are present in the mouse and rat nervous system, RACE, RT-PCR, and RNase protection analyses of Gfra4 transcripts showed that majority of these transcripts encode truncated protein isoforms without a functional signal sequence (5) (data not shown). Taken together, our results strongly suggest that the Gfra4 transcripts seen by Masure et al. (27) in the rat brain encode unfunctional protein isoforms.

GFRalpha 4 Is the Functional Receptor for PSPN-- The binding and cross-linking studies demonstrated that association of PSPN with the Ret receptor protein-tyrosine kinase is mediated by GFRalpha 4. Ligand displacement binding showed that in the presence of Ret, only PSPN but not GDNF, NRTN, or ARTN was effective in displacing 125I-PSPN from GFRA4-expressing Neuro-2a cells. The dissociation constant (Kd) of about 100 pM found here is 10 times lower than the Kd of about 1 nM reported for mouse PSPN, binding to chicken GFRalpha 4 (3), and 60 times lower than the about 6 nM reported for rat PSPN binding to immobilized rat GFRalpha 4 fusion protein (27), but similar to those reported for GFRalpha 1, GFRalpha 2, and GFRalpha 3 and their cognate ligands (28-30). The lower binding of mammalian PSPN to chicken GFRalpha 4 probably reflects the species difference in GFRalpha 4 structure. PSPN has not yet been characterized from chicken and, if it exists, might differ significantly from the mammalian PSPN.

The cross-linking studies show that PSPN is able to bind GFRalpha 4 also in the absence of Ret. This is consistent with the model that a GDNF family ligand first binds to the corresponding GFRalpha receptor and subsequently the ligand-GFRalpha complex binds to Ret (31). However, our results also agree with the alternative model in which the ligand binds a preformed GFRalpha -Ret complex (32). Although PSPN binding to chicken GFRalpha 4 has been shown earlier (3), we demonstrate here for the first time that the association of PSPN with GFRalpha 4 results in activation of the Ret tyrosine kinase. PSPN treatment of cells expressing GFRalpha 4 rapidly induced Ret autophosphorylation in a dose-dependent manner. PSPN is unable to stimulate Ret autophosphorylation in cells that do not express GFRalpha 4. Thus, the GFRalpha 4a isoform characterized in this report is a functional receptor for PSPN in triggering Ret activation. In contrast to this, the rat GFRalpha 4 isoform with a poor signal sequence described by Masure et al. (27) bound PSPN only as soluble fusion protein and did not lead to Ret activation.

We also demonstrate that PSPN but not other GDNF family ligands, can promote the survival of cultured SCG neurons expressing Gfra4 and RET. In our assay, neurons coexpressing RET plus GFRalpha 4 showed an elevated response to GDNF and NRTN but not to ARTN, however, also the uninjected neurons responded to GDNF and NRTN. We could detect Ret, as well as Gfra1 and Gfra2, but not Gfra4 transcripts in mouse SCG (not shown). Therefore, it seems clear that GDNF and NRTN maintain these neurons via activation of endogenous receptors but not via GFRalpha 4. Absence of Ret in mouse SCG, reported in a previous study (33), probably results from different mouse strains used. Taken together, the binding of PSPN to GFRalpha 4-Ret complex triggers functional cellular responses.

Putative Role for GFRalpha 4 in Medullary Thyroid Carcinoma-- As the RET proto-oncogene plays an important role in the oncogenesis of MTC, a logical step was to examine GFRalpha 4 expression in these tumors. GFRA4 transcripts encoding the two GPI-anchored isoforms were expressed at high level in all MTC samples. In addition, moderate levels of GFRA4c encoding the putative soluble form of GFRalpha 4 were found in all MTC samples. In contrast, no GFRA4 expression was detected in any other type of thyroid tumor analyzed. Taken together, our data suggest that the expression of GFRA4 in thyroid tumors is C-cell specific, and therefore antibodies against GFRalpha 4 could be useful in tumor diagnostics. It will be important to study whether metastases derived from MTC tumors and other endocrine tumors such as pheochromocytomas express GFRA4.

Strong RET expression was also localized to the malignant C-cells consistent with previous reports (25, 34). Low levels of GFRA1, GFRA2, and GFRA3 transcripts were present in subsets of tumor cells, indicating that their expression is not specific to particular thyroid tumor cell type. This result is in line with a recent report showing GFRA1 and GFRA2 expression in some MTC tumor cells (35). However, our results showing low GFRA1 and GFRA2 expression also in normal thyroid, in other thyroid tumor types, and not in all MTC samples, does not support the idea that GFRalpha 1 or GFRalpha 2 play a role in the primary pathogenesis of MTC. We have proposed that coexpression of GFRalpha 4 with mutated RET may specify tissues affected by MEN2 syndromes (5), and thus GFRalpha 4 be necessary or at least permissive for the initial hyperplasia of C-cells occurring in MTC. On the other hand, as suggested in a recent report, other GFRalpha receptors could interfere with the dimerization of mutant RET receptors, and therefore inhibit tumor development in tissues not involved in MEN2 syndromes (36). It should be possible to test these hypotheses by examining whether the tumor development is suppressed or enhanced in different GFRalpha -deficient mice crossed with MEN2 transgenic mice (17-19). Further in vitro studies are also needed to reveal the roles for PSPN signaling via different GFRalpha 4 isoforms through normal and mutant RET.

    ACKNOWLEDGEMENTS

We thank Eila Kujamäki, Maarit Ohranen, and Satu Åkerberg for excellent technical assistance, Drs. J. Milbrandt and E. M. Johnson, Jr., for ARTN, and Dr. M. Billaud for the human RET construct.

    FOOTNOTES

* This work was supported in part by the Academy of Finland, Biocentrum Helsinki, Cephalon, Inc., EU Biomed II Grant BMH4-97-2157, and the Sigrid Juselius Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ291673, AJ291674, and AJ291675.

§ Contributed equally to the results of this article.

** Biocentrum Helsinki fellow. To whom correspondence should be addressed. Tel: 358-9-191-59-359; Fax: 358-9-191-59-366; E-mail: Saarma@operoni.helsinki.fi.

Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008279200

2 M. Lindahl and T. Timmusk, unpublished data.

    ABBREVIATIONS

The abbreviations used are: GDNF, glial cell line-derived neurotrophic factor; ARTN, artemin; FTA, follicular thyroid adenoma; FTC, follicular thyroid carcinoma; GFR, glial cell line-derived neurotrophic factor family receptor; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; NRTN, neurturin; PSPN, persephin; PTC, papillary thyroid carcinoma, SCG, superior cervical ganglion; PSPN, persephin; GPI, glycosylphosphatidylinositol; RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends; NGF, nerve growth factor; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Airaksinen, M. S., Titievsky, A., and Saarma, M. (1999) Mol. Cell. Neurosci. 13, 313-325[CrossRef][Medline] [Order article via Infotrieve]
2. Baloh, R. H., Enomoto, H., Johnson, E. M., Jr., and Milbrandt, J. (2000) Curr. Opin. Neurobiol. 10, 103-110[CrossRef][Medline] [Order article via Infotrieve]
3. Enokido, Y., de Sauvage, F., Hongo, J.-A., Ninkina, N., Rosenthal, A., Buchman, V. L., and Davies, A. M. (1998) Curr. Biol. 8, 1019-1022[Medline] [Order article via Infotrieve]
4. Thompson, J., Doxakis, E., Piñon, L. G. P., Strachan, P., Buj-Bello, A., Wyatt, S., Buchman, V. L., and Davies, A. M. (1998) Mol. Cell. Neurosci. 11, 117-126[CrossRef][Medline] [Order article via Infotrieve]
5. Lindahl, M., Timmusk, T., Rossi, J., Saarma, M., and Airaksinen, M. S. (2000) Mol. Cell. Neurosci. 15, 522-533[CrossRef][Medline] [Order article via Infotrieve]
6. Milbrandt, J., de Sauvage, F. J., Fahrner, T. J., Baloh, R. H., Leitner, M. L., Tansey, M. G., Lampe, P. A., Heuckeroth, R. O., Kotzbauer, P. T., Simburger, K. S., Golden, J. P., Davies, J. A., Vejsada, R., Kato, A. C., Hynes, M., Sherman, D, Nishimura, M, Wang, L. C., Vandlen, R., Mottat, B., Klein, R. D., Poulson, K., Gray, C., Garces, A., Henderson, C, E., Phillips, H, S., and Johnson, E. M., Jr. (1998) Neuron 20, 245-253[Medline] [Order article via Infotrieve]
7. Jaszai, J., Farkas, L., Galter, D., Reuss, B., Strelau, J., Unsicker, K., and Kriegelstein, K. (1998) J. Neurosci. Res. 53, 494-501[CrossRef][Medline] [Order article via Infotrieve]
8. Ho, T. W., Bristol, L. A., Coccia, C., Li, Y., Milbrandt, J., Johnson, E. M., Jr., Jin, L., Bar-Peled, O., Griffin, J. W., and Rothstein, J. D. (2000) Exp. Neurol. 161, 664-675[CrossRef][Medline] [Order article via Infotrieve]
9. Edery, P., Eng, C., Munnich, A., and Lyonnet, S. (1997) BioEssays 19, 389-395[Medline] [Order article via Infotrieve]
10. Eng, C. (1999) J. Clin. Oncol. 17, 380-393[Abstract/Free Full Text]
11. Ponder, B. A. (1999) Cancer Res. 59, 1736s-1741s[Medline] [Order article via Infotrieve]
12. Tsuzuki, T., Takahashi, M., Asai, N., Iwashita, T., Matsuyama, M., and Asai, J. (1995) Oncogene 10, 191-198[Medline] [Order article via Infotrieve]
13. Santoro, M., Carlomagno, F., Romanova, A., Bottaro, D. L. P., Dathan, N. A., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. H., and Di Fiore, P. P. (1995) Science 267, 381-383[Medline] [Order article via Infotrieve]
14. Iwashita, T., Asai, N., Murakami, H., Matsuyama, M., and Takahashi, M. (1996) Oncogene 12, 481-487[Medline] [Order article via Infotrieve]
15. Carlomagno, F., Melillo, R. M., Visconti, R., Salvatore, G., de Vita, G., Lupoli, G., Yu, Y., Jing, S., Vecchio, G., Fusco, A., and Santoro, M. (1998) Endocrinology 139, 3613-3619[Abstract/Free Full Text]
16. Bongarzone, I., Vigano, E., Alberti, L., Borrello, M. G., Pasini, B., Greco, A., Mondellini, P., Smith, D. P., Ponder, B. A. J., Romeo, G., and Pierotti, M. A. (1998) Oncogene 16, 2295-2301[CrossRef][Medline] [Order article via Infotrieve]
17. Michiels, F.-M., Chappuis, S., Caillou, B., Pasini, A., Talbot, M., Monier, R., Lenoir, G. M., Feunteun, J., and Billaud, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3330-3335[Abstract/Free Full Text]
18. Smith-Hicks, C. L., Sizer, K. C., Powers, J. F., Tischler, A. S., and Constantini, F. (2000) EMBO J. 19, 612-622[Abstract/Free Full Text]
19. Acton, D. S., Velthuyzen, D., Lips, C. J. M., and Hoppener, J. W. M. (2000) Oncogene 19, 3121-3125[CrossRef][Medline] [Order article via Infotrieve]
20. Laurikainen, A., Hiltunen, J. O., Thomas-Crusells, J., Vanhatalo, S., Arumäe, U., Airaksinen, M. S., Klinge, E., and Saarma, M. (2000) J. Neurobiol. 43, 198-205[CrossRef][Medline] [Order article via Infotrieve]
21. McGonigle, P., and Molinoff, P. B. (1994) in Basic Neurochemistry (Siegel, G. J., ed), 5th Ed. , pp. 209-230, Raven Press, New York
22. Rossi, J., Luukko, K., Poteryaev, D., Laurikainen, A., Sun, Y.-F., Laakso, T., Eerikäinen, S., Tuominen, R., Lakso, M., Rauvala, H., Arumäe, U., Saarma, M., and Airaksinen, M. S. (1999) Neuron 22, 243-252[Medline] [Order article via Infotrieve]
23. Udenfried, S., and Kodukula, K. (1995) Annu. Rev. Biochem. 64, 563-591[CrossRef][Medline] [Order article via Infotrieve]
24. Lou, H., and Gagel, R. F. (1998) J. Endocrinol. 156, 401-405[Abstract/Free Full Text]
25. Nakamura, T., Ishizaka, Y., Nagao, M., Hara, M., and Ishikawa, T. (1994) J. Pathol. 172, 255-260[Medline] [Order article via Infotrieve]
26. Lorenzo, M. J., Eng, C., Mulligan, L. M., Stonehouse, T. J., Healey, C. S., Ponder, B. A. J., and Smith, D. P. (1995) Oncogene 10, 1377-1383[Medline] [Order article via Infotrieve]
27. Masure, S., Cik, M., Hoefnagel, E., Nosrat, C. A., Van der Linden, I., Scott, R., Van Gompel, P., Lesage, A. S. J., Verhasselt, P., Ibáñez, C. F., and Gordon, R. D. (2000) J. Biol. Chem. 275, 39427-39434[Abstract/Free Full Text]
28. Klein, R. D., Sherman, D., Ho, W-H., Stone, D., Bennett, G. L., Moffat, B., Vandlen, R., Simmons, L., Gu, Q., Hongo, J-A., Devaux, B., Poulsen, K., Armanini, M., Nozaki, C., Asai, N., Goddard, A., Phillips, H., Henderson, C., E., Takahashi, M., and Rosenthal, A. (1997) Nature 387, 717-721[CrossRef][Medline] [Order article via Infotrieve]
29. Trupp, M., Raynoschek, C., Belluardo, N., and Ibáñez, C. F. (1998) Mol. Cell. Neurosci. 11, 47-63[CrossRef][Medline] [Order article via Infotrieve]
30. Baloh, R. H., Tansey, M. G., Lampe, P. A., Fahrner, T. J., Enomoto, H., Simburger, K. S., Leitner, M. L., Araki, T., Johnson, E. M., Jr., and Milbrandt, J. (1998) Neuron 21, 1291-1302[Medline] [Order article via Infotrieve]
31. Jing, S., Wen, D., Yu, Y., Holst, P. L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J. -C., Hu, S., Altrock, B. W., and Fox, G. M. (1996) Cell 85, 1113-1124[Medline] [Order article via Infotrieve]
32. Sanicola, M., Hession, C., Worley, D., Carmillo, P., Ehrenfels, C., Walus, L., Robinson, S., Jaworski, G., Wei, H., Tizard, R., Whitty, A., Pepinsky, R. D., and Cate, R. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6238-6243[Abstract/Free Full Text]
33. Buj-Bello, A., Adu, J., Piñon, L. G. P., Horton, A., Thompson, J., Rosenthal, A., Chinchetru, M., Buchman, V. L., and Davies, A. M. (1997) Nature 387, 721-724[CrossRef][Medline] [Order article via Infotrieve]
34. Santoro, M., Rosati, R., Grieco, M., Berlingieri, M. T., D'Amato, G. L.-C., de Fransiscis, V., and Fusco, A. (1990) Oncogene 5, 1595-1598[Medline] [Order article via Infotrieve]
35. Frisk, T., Farnebo, F., Zedenius, J., Grimelius, L., Hoog, A., Wallin, G., and Larsson, C. (2000) Eur. J. Endocrinol. 142, 643-649[Medline] [Order article via Infotrieve]
36. Kawai, K., Iwashita, T., Murakami, H., Hiraiwa, N., Yoshiki, A., Kusakabe, M., Ono, K., Iida, K., Nakayama, A., and Takahashi, M. (2000) Cancer Res. 60, 5254-5260[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.