Genomic structure and chromosomal localization of the ... - Nature

Oncogene (1998) 16, 597 ± 601  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

Genomic structure and chromosomal localization of the human GDNFR-a gene Charis Eng1,2, Shirley M Myers3, Michael D Kogon3, Michele Sanicola4, Cathy Hession4, Richard L Cate4 and Lois M Mulligan3 1

Dana-Farber Cancer Institute, Charles A Dana Human Cancer Genetics Unit Richard and Sara Smith Laboratories, SM822 1 Jimmy Fund Way, Boston, Massachusetts 02115, USA; 2CRC Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ, UK; 3Departments of Pathology and Paediatrics, Queen's University, Kingston, Ontario K7L 3N6, Canada; 4 Department of Molecular Biology, Biogen, Inc. Cambridge Massachusetts 02142, USA.

GDNFR-a is a glycosyl-phosphotidylinositol-linked receptor for glial cell line-derived neurotrophic factor (GDNF). GDNF binds to GDNFR-a and this complex, in turn, is believed to interact with the RET receptor tyrosine kinase to eect downstream signalling. GDNFR-a belongs to a novel gene family without strong homology to known genes. Thus, little information has been available to help predict genomic structure or location of this gene. In this study, the genomic organization of human GDNFR-a was delineated through a combination of PAC clone characterization, long distance PCR and sequence analyses. Exon-intron boundaries were de®ned by comparing the size and sequence of the genomic PCR products to those predicted by the cDNA sequence. The human GDNFRa gene comprises 9 exons. GDNFR-a PAC clones were used for FISH analysis to map this gene to 10q26. Keywords: RET; GFRa-1; GDNF; Exon ± Intron; 10q

Introduction Glial cell line-derived neurotrophic factor receptor alpha (GDNFR-a) is a recently identi®ed glycosylphosphotidylinositol (GPI)-linked cell surface molecule primarily expressed in the developing kidney and the nervous system. The human GDNFR-a gene has recently been isolated and encodes an open reading frame of H60 of 465 amino acids with a signal peptide at the amino terminus, a stretch of 20 ± 23 hydrophobic amino acids at the carboxy terminus and three possible glycosylation sites (Jing et al., 1996; Treanor et al., 1996; Sanicola et al., 1997). Although GDNFR-a has no strong homologies to any other receptor, it does encode 30 cysteine residues with similar spacing and arrangement to those found in many cytokine receptors (Treanor et al., 1996; Sanicola et al., 1997). The absence of any C-terminal hydrophilic sequence and the presence of a cluster of small amino acids that de®ne a site for the GPI-linkage suggest that GDNFRa is a glycolipid-linked extracellular protein attached to the cell membrane, without an intracellular signalling domain.

GDNFR-a is a component of the GDNF/GDNFRa/RET signalling complex. GDNF binds with high anity to GDNFR-a to form a membrane-bound, non-signalling complex, postulated to be a heterotetramer (Jing et al., 1996). This ligand complex, in turn, interacts with and activates the RET receptor tyrosine kinase. RET is expressed in an array of tissues and tumours, chie¯y those derived from the neural crest and the developing kidney (Takahashi et al., 1985, 1988; Santoro et al., 1990; Pachnis et al., 1993; Nakamura et al., 1994; Durbec et al., 1996). RET activation plays a major role in growth survival and/or dierentiation of neural crest derivatives and also in induction of the metanephric kidney. Consistent with this, RET-/- and GDNF-/- mice lack enteric ganglia and have dysgenic or agenic kidneys (Schuchardt et al., 1994; Moore et al., 1996; Pichel et al., 1996; SaÂnchez et al., 1996). Thus, GDNFR-a, as a member of the RET ligand complex, is also predicted to contribute to these developmental processes. Germline mutations of the RET proto-oncogene are found in the majority of cases of multiple endocrine neoplasia type 2 (MEN 2), an autosomal dominant syndrome classically characterized by medullary thyroid carcinoma (MTC), phaeochromocytoma (phaeo) and hyperparathyroidism (Mulligan et al., 1993, 1994; Donis-Keller et al., 1993; Carlson et al., 1994; Eng et al., 1994, 1995, 1996; Hofstra et al., 1994; Bolino et al., 1995). Somatic activating RET mutations have also been found in sporadic MTC and phaeochromocytoma (reviewed in Eng and Mulligan, 1997). Loss of function mutations of RET have been implicated in Hirschsprung disease (HSCR), a developmental disorder characterized by absence of enteric ganglia (reviewed in Eng and Mulligan, 1997). Whether GDNFR-a mutations might also contribute to these phenotypes has not been investigated. The human GDNFR-a locus has not been well characterized. In this study, we have delineated the exon ± intron boundaries of this gene using a combination of PAC clone characterization and inter-exon PCR. We have also mapped GDNFR-a to 10q26.

Results GDNFR-a Exon ± Intron boundaries

Correspondence: C Eng and LM Mulligan Received 23 July 1997; accepted 17 September 1997

The exon ± intron boundaries of GDNFR-a were identi®ed using a combination of long distance and regular PCR. Forward and reverse primers were

Human GDNFR-a genomic structure C Eng et al

598

selected using the cDNA sequence of GDNFR-a (Sanicola et al., 1997; Genbank U97144). We compared the size of ampli®cation products; obtained using genomic DNA and ®rst strand cDNA from the GDNFR-a-expressing TT cell line as templates. Larger PCR product sizes obtained with genomic templates, compared to cDNA templates, were suggestive of the presence of introns located in the region de®ned by the ¯anking primers. Bidirectional sequencing of these products was used to identify the position of the exon ± intron boundaries and the contiguous intronic sequence (Table 1). Using this strategy, 9 exons and 8 introns were identi®ed (Table 1, Figure 1). Two of the GDNFR-a introns were too large to be ampli®ed by regular or long distance PCR. PCR fragments from exon 1 and exon 4 were used to isolate PAC clones 219C19 and 202A8, respectively (S Scherer, CGAT Genome Resource Facility, Toronto, ON, Canada). PAC 202A8 was subcloned using HindIII. Subclones containing exons 4,5,7 or 8 were isolated using exon-speci®c products as probes. The exon ± intron boundaries ¯anking introns 4 and 7 (Table 1) were characterised by sequencing the selected subclones using exon-speci®c primers. FISH analysis PAC clone 219C19 was labelled and mapped by FISH (B Beatty, CGAT FISH Mapping Facility, Toronto, ON, Canada). The clone was regionally assigned to proximal chromosome sub-band 10q26 (Figure 2) by analysis of 20 well-spaced metaphases.

Discussion GDNFR-a encodes an extracellular protein attached to the cell surface by a GPI-linkage. It shows little homology to other known protein families although it contains 30 cysteine residues spaced similarly to those in the cytokine receptors (Treanor et al., 1996). We have shown that GDNFR-a comprises 9 coding exons ranging in size from 40 ± 337 bp. The structural organization of this gene is unlike that of other cytokine receptors. A signal peptide is encoded in exons 1 and 2, and a small cluster of 20 ± 23 hydrophobic amino acids, near the Cterminus, encoded in exon 9. No other structural motifs are identi®ed. Interestingly, an insertion of ®ve amino acids, reported in some studies (Jing et al., 1996; Treanor et al., 1996), was not found in this analysis. These amino acids are predicted to lie at the boundary of exons 3 and 4. However, the relevant codons were not identi®ed in intron 3 either directly downstream of exon 3 or upstream of exon 4 (Table 1). Further sequencing of RT ± PCR products spanning these regions did not identify this sequence. Several studies have shown that GDNFR-a is a member of a family of proteins with similar structures. A recently identi®ed family member, NTNR-a (TrnR2, RETL2) has 48% homology to GDNFR-a (Baloh et al., 1997; Buj-Bello et al., 1997; Klein et al., 1997; Sanicola et al., 1997). This homology is particularly strong in the regions between GDNFR-a amino acids 117 and 262 which are encoded within exons 3, 4 and 5. There is an overall 74% identity across this stretch of 128 amino acids, perhaps

Table 1 Exon-intron boundaries in GDNFR-a Exon no. 1 2 3 4 5 6 7 8 9

Exon Size* (bp)

Basepair No.

Sequence

40 294 84 337 110 135 182 54 147

1 ± 40 41 ± 334 335 ± 418 419 ± 755 756 ± 865 866 ± 1000 1001 ± 1182 1183 ± 1236 1237 ± 1384

ATGTTCCTGG . . . CCGCTCTTGGgtaagtcgag tgccccgcagACTTGCTCCT . . . AGCCTGCAGGgtacggtgac ttgtttttagGAAATGATCT . . . TTCATATCAGgtaagcagat gcatttgcagTGGAGCACAT . . . ACATCTGCAGgtaagtgggc cctattgtagATCTCGCCTT . . . GGGCTTATTGgtaagatccg attcttttagGCACAGTCAT . . . ACATGTCTTAgtgagnttgt tttactgcagAAAATGCAAT . . . AAATTTACAGgtgagaactg ccctctccagGCACAGAAGC . . . TATTTCCAATgtaagtatgt ttccttacagGGTAATTATG . . . AACATCATAG

*Coding regions only. For exons 1 and 9, sizes refer to coding portions only, excluding 5' and 3' UTRs, respectively

Figure 1 Schematic representation of the genomic structure of human GDNFR-a. Coding exon sizes are on top while primers (represented by arrowheads) used to delineate genomic structure and intronic sequence are below. Sizes of introns predicted from PCR analysis is indicated below the exon ± intron map. N/A, intron size not determined


599

Chromosome 10

Figure 2 FISH demonstrates that GDNFR-a-containing clones speci®cally localize to 10q26. See text

suggesting a common functional domain shared by these proteins. This is not entirely surprising as both these molecules bind GDNF and RET. Preliminary evidence suggests that GDNFR-a and NTNR-a bind GDNF and participate in ligand complexes, which activate RET. Additionally, NTNR-a also interacts with a GDNF-like molecule, neurturin (NTN) to activate RET. The delineation of the genomic structure of GDNFR-a should greatly facilitate similar determination of exon ± intron boundaries for NTNR-a and perhaps other genes encoding proteins belonging to this novel family. Materials and methods TT cell line The human medullary thyroid carcinoma cell line (Leong et al., 1981) was purchased from the American Type Culture Collection (Rockville, MD, USA) and grown according to their recommendations. TT cells were grown as monolayers and replated weekly. These cells were treated with alltrans-retinoic acid 24 ± 48 h before harvesting to increase the levels of GDNFR-a transcripts. Template DNA Genomic DNA was isolated from anonymised peripheral blood leucocytes using standard methods or purchased from Promega (Madison, WI, USA). Total RNA was extracted from the TT cell line using TRIzol reagent (Life Technologies, Burlington, ON, Canada and Gaithersburg, MD, USA). One mg of RNA in 10 ml of DEPC-treated H2O was heated to 708C for 5 min and then quenched on ice brie¯y. First strand cDNA was obtained by incubating the template RNA in 50 mM Tris-HCl pH 8.3, 8 mM MgCl2, 30 mM KCl, 10 mM DTT, 2 mM each dNTPs,

2 mM oligo(dT)15 or random hexamers and 5 U AMV reverse transcriptase (Promega) at 428C for 1 h. First strand cDNA served as template for RT ± PCR. Long distance PCR Long distance PCR was performed according to the manufacturer's recommendation (TaKaRa LA PCR Kit, Ver. 2, Shiga, Japan). In brief, 200 ± 400 ng of genomic DNA was used as template in a 50 ml reaction volume. Final concentrations of reagents include MgCl2 at 1.5 mM, dNTPs each at 400 mM and 0.2 mM of each primer. PCR cycling began with a hot start of 948C 6 1 min, the addition of 0.5 units of Hi IQ Taq Pol (TaKaRa), followed by 948C 6 2 min. Subsequently, PCR was performed using 14 cycles of 988C 6 20 s ? 64 or 688C 6 20 min, 15 cycles of 988C 6 20 s ? 688C 6 20 min (with increments of 15 s per cycle) and a ®nal chain extension step of 728C 6 10 min. PCR products were fractionated on 1% LMP agarose (BioRad, Hercules, CA, USA or Fisher, Pittsburgh, PA, USA), column puri®ed (Wizard PCR Preps, Promega) with elution in water or 1 6 TE8 heated to 908C and subjected to sequence analysis. RT ± PCR and regular PCR of genomic DNA PCR was carried out using a sense and antisense primer pair in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.75 ± 1.75 mM MgCl2, 0.01% gelatin, 1 mM of each primer, 200 mM each of dNTPs and 1.5 U Taq DNA Pol (Life Technologies) with 40 cycles of 958C 6 1 min, 538 or 558C 6 1 min and 728C 6 1 min, followed by a ®nal chain extension at 728C 6 10 min. If the Perkin ± Elmer PCR kit (Perkin ± Elmer Corp, Norwalk, CT, USA) was used, cycling conditions were identical except for annealing at 598C. All PCR products were gel and column puri®ed (Wizard PCR preps, Promega) as described above.


600

Primers

Sequence analysis

Primers used for delineation of GDNFR-a intron ± exon boundaries were derived from sequence from GenBank U97144. The ®rst base of the initiator codon is de®ned as bp 1 in the primers below:

100 ± 600 ng of PCR product was used as sequencing template. In general, 500 ± 600 ng of long PCR product was required as template. PCR products were subjected to cycle sequencing using the DTaq Ver.2.0 cycle sequencing kit (Amersham Life Sciences, Arlington Heights, IL, USA) either with the incorporation of [a-35S]dATP[a-32P]dCTP according to the manufacturer's recommendations or by ¯uorometric methods on the ABI373A automated sequencer and the DyeDeoxyTerminator Cycle Sequencing Kit (ABI, Perkin-Elmer Corp) as previously described (Ivanchuk et al., 1996; Liaw et al., 1997).

HRAF1

5'-ATGTTCCTGGCGACCCTGT -3' (exon 1; bp 1 ± 18);

RAR8

5'-AGTTGGTCTCCTTGCCCGC -3' (exon 2; bp 178 ± 160);

HRAR2

5'-CTGGTACATGCTCCAGTA -3' (exon 2; bp 324 ± 307);

RAF7

5'-CGCACGCTAAGGCAGTGCGT -3' (exon 2; bp 139 ± 158);

RAF12

5'-TTTACTGGAGCATGTACC -3' (exon 2; bp 305 ± 322);

GRA2F

5'-ATGAACCAGTTAACAGCAGATTGTCA- 3' (exon 3, bp 362 ± 387)

RAR14

5'-ATATGAATGGGACCACCCGG -3' (Exon 3; bp 415 ± 396);

RAF11

5'-GCAACCTCGACGACATTTGC -3' (exon 4; bp 467 ± 486);

RAR19

5'-CATAGGAGCACACAGGCACG -3' (exon 4; bp 691 ± 672);

RAF19

5'-ATCGTGCCTGTGTGCTCCTA -3' (exon 4; bp 670 ± 689);

GRA19R

5'-CTCCCTCTCTTCATAGGAGCACTC -3' (exon 4; bp 702 ± 679);

RAF5B

5'-TACCAACTGCCAGCCAGAGT -3' (exon 5; bp 777 ± 796);

GRA11F

5'-GCTGTCTAAAGGAAAACTACGCTGACT -3' (exon 5; bp 812 ± 838);

RAR16

5'-GAAAGAACGCAGGTATATGC -3' (intron 5);

GRA15F

5'-CTACATAGACTCCAGTAGCCTCAGTG -3' (exon 6; bp 885 ± 910);

GRA12R

5'-AAAATTTCAAGCACTCTTCTAGGTCG -3' (exon 6; bp 945 ± 970);

RAF25

PAC clone characterization and FISH analysis PCR products corresponding to exons 1 and 4 were used to screen a PAC library, resulting in the isolation of two PAC clones, 219C19 and 202A8, respectively (S Scherer, CGAT Genome Resource Facility, Toronto, Canada). Using PAC clone 219C19 as a probe, FISH was performed on normal human peripheral leucocyte chromosomes counterstained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-¯uorescein isothiocyanate (FITC). Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometrics, Tucson, AZ, USA). Separate images of DAPI banded chromosomes and of FITC targeted chromosomes were obtained. Hybridization signals were acquired, pseudo coloured blue (DAPI) or yellow (FITC), overlaid electronically and merged using Adobe Photoshop 3.0 software. In order to identify the boundaries of introns 4 and 7, PAC clone 202A8 was subcloned for further analysis. PAC DNA was digested with HindIII, ligated into pBluescript vector, and the constructs transfected into electrocompetent DH5a E. coli using standard methods (Ausubel et al., 1995). Colonies containing inserts were selected and gridded onto LB agar plates containing ampicillin. Colony lift membranes were prepared using Hybond N+ membrane (Amersham-Life Sciences) according to standard protocols (Ausubel et al., 1995). Membranes were prehybridized at 658C using standard conditions and hybridized to 32P-labeled exonspeci®c PCR products. Membranes were washed with 1 6 SSC, 0.1% SDS at 658C and exposed to X-ray ®lm (XAR-5, Kodak, Rochester, NY, USA) for 10 to 30 min at 7708C.

5'-ATTTCCTCCTGTTTTACTGC -3' (intron 6);

GRA13R

5'-GGTAGTGGCAGTGGTGGTCTGTA -3' (exon 7; bp 1086 ± 1064);

GRA16R

5'-GGCAAAACATGAGTGGGAATTTCA -3' (exon 7; bp 1163 ± 1140);

RAF10

5'-CAGGCACAGAAGCTGAAATC -3' (exon 8; bp 1180 ± 1199);

RAR13

5'-TCTATAAATGCACGAAGCCT -3' (intron 8);

RAR9

5'-GCAGCTATGATGTTTCTGTT -3' (exon 9; bp 1387 ± 1368);

GRA20R

5'-TTTTTCATGTCCATATTGTATTTTT -3' (3'UTR; bp 1417 ± 1393).

Acknowledgements We thank D Croaker, P Dahia, A GoÈssling, S Ivanchuk, M Linder, D Marsh and Z Zheng for helpful discussions, assistance and/or critical review of the manuscript, B Beatty for FISH analysis and S Scherer for PAC clone isolation. This work was supported by grants from the Medical Research Council of Canada (LMM), the Kingston General Hospital and Hospital for Sick Children Foundations (LMM) and the Clare Nelson Bequest (LMM); the Lawrence and Susan Marx Investigatorship in Human Cancer Genetics (CE), a Young Investigator Award from the Markey Charitable Trust (CE), the Charles A Dana Foundation (CE) and a Patterson Fellowship (CE).


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