GFRa-4, a New GDNF Family Receptor

Jane Thompson, Epaminondas Doxakis, Luzia G. P. Pinon,Philip Strachan, Anna Buj-Bello,1 Sean Wyatt,Vladimir L. Buchman, and Alun M. Davies2

School of Biomedical Sciences, University of St. Andrews, Bute Medical Buildings,St. Andrews, Fife KY16 9TS, Scotland

GFRa-1, GFRa-2, and GFRa-3 constitute a family of struc-turally related, glycosyl-phosphatidylinosital-linked, cellsurface proteins, two of which, GFR a-1 and GFRa-2, arecomponents of the receptor complex for the neurotrophicfactors GDNF and neurturin, respectively. By screening anembryonic chicken brain cDNA library with a GFR a-1probe at low stringency, we isolated cDNAs encoding anadditional member of the GFR a family, GFR a-4. The nucle-otide sequence predicts a 431-amino-acid secreted pro-tein that is more closely related to GFR a-1 and GFRa-2than to GFR a-3. GFRa-4 mRNA is expressed in distinctivepatterns in the brain and several other organs and tissuesof the chicken embryo. Our findings extend the family ofGFRa proteins and provide information about the tissuesin which GFR a-4 may function during development.

INTRODUCTION

GDNF is a distantly related member of the transform-ing growth factor-b family that was isolated from a glialcell line (Lin et al., 1993). It is a potent survival factor forcultured midbrain dopaminergic neurons (Lin, 1993)and motoneurons (Henderson et al., 1994; Zurn et al.,1994; Li et al., 1995; Oppenheim et al., 1995; Yan et al.,1995; Houenou et al., 1996) and protects midbraindopaminergic neurons from MPTP toxicity (Tomac et al.,1995), 6-hydroxydopamine toxicity (Bowenkamp et al.,1996; Choi-Lundberg et al., 1997; Kearns et al., 1997), andaxotomy-induced degeneration (Beck et al., 1995; Lu andHagg, 1997). GDNF also promotes the survival ofseveral other populations of CNS neurons, includingnoradrenergic neurons of the locus coeruleus (Arenas et

al., 1995), Purkinjie cells (Mount et al., 1995) and basalforebrain cholinergic neurons (Ha et al., 1996; Williamset al., 1996). Accordingly, GDNF is widely expressed inthe brain (Schaar et al., 1993; Stromberg et al., 1993;Springer et al., 1994; Arenas et al., 1995; Nosrat et al.,1997; Pochon et al., 1997; Trupp et al., 1997). In theperipheral nervous system, GDNF promotes the in vitrosurvival of embryonic sympathetic, parasympathetic,and sensory neurons and is expressed in a wide varietyof tissues during development (Suter-Crazzolara andUnsicker, 1994; Buj-Bello et al., 1995; Trupp et al., 1995;Wright and Snider, 1996). The physiological relevance ofmany of the in vitro effects of GDNF on neuronalsurvival have been substantiated by the finding of anappreciable reduction in the number of sensory, motor,sympathetic, and enteric neurons in GDNF2/2 mice(Moore et al., 1996; Pichel et al., 1996; Sanchez et al.,1996). The kidney also fails to develop in GDNF2/2

mice, indicating a role for GDNF in kidney developmentwhich accords with the expression of GDNF in thisorgan during development (Suter-Crazzolara and Un-sicker, 1994; Buj-Bello et al., 1995; Trupp et al., 1995;Nosrat et al., 1997). Neurturin is a recently identifiedhomologue of GDNF that promotes the survival ofcultured rat superior cervical sympathetic, dorsal root,and nodose ganglion neurons (Kotzbauer et al., 1996).

GDNF and neurturin signal via multicomponent re-ceptors that consist of the Ret receptor tyrosinekinase plus one of two structurally related glycosyl-phosphatidylinositol (GPI)-linked receptors (Durbec etal., 1996; Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1996;Vega et al., 1996; Worby et al., 1996; Baloh et al., 1997; Buj-Bello et al., 1997; Creedon et al., 1997; Jing et al., 1997;Klein et al., 1997; Sanicola et al., 1997; Suvanto et al., 1997;Widenfalk et al., 1997). Although originally described byseveral different names, these two GPI-linked receptorsare now termed GFRa-1 and GFRa-2 (GDNF Receptor

1 Present address: Equipe Genetique Humaine, Institut de Genet-ique et de Biologie Moleculaire, 1, rue Laurent Fries, B.P. 163, 67404Illkirch Cedex, France.

2 To whom correspondence should be addressed. E-mail: amd2@st-and.ac.uk.

MCNMolecular and Cellular Neuroscience 11, 117–126 (1998)

Article No. CN980682

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1044-7431/98 $25.00Copyright r 1998 by Academic PressAll rights of reproduction in any form reserved.

Nomenclature Committee, 1997). Although Ret activa-tion and signaling by GDNF and neurturin require oneof these GPI-linked receptors, there is some controversyover the specificity of GFRa-1 and GFRa-2 receptors forthese ligands. Equilibrium and competition bindingstudies (Klein et al., 1997) and the survival responses ofneurons ectopically coexpressing Ret plus GFRa-1 orGFRa-2 (Buj-Bello et al., 1997) have shown that GFRa-1is a specific high-affinity receptor for GDNF and thatGFRa-2 is a specific high-affinity receptor for neurturin.Although studies of Ret phosphorylation in fibroblastsectopically expressing Ret plus GFRa-1 or GFRa-2 suggestthat the Ret/GFRa-2 complex is a preferential receptor forneurturin (Baloh et al., 1997), the Ret/GFRa-1 complexis equally effective in mediating responses to GDNF andneurturin (Baloh et al., 1997; Creedon et al., 1997).Furthermore, GDNF has been shown to promote Retphosphorylation in COS cells coexpressing Ret pluseither GFRa-1 or GFRa-2 (Suvanto et al., 1997). Al-though these studies in fibroblast and COS cell lineshave shown a degree of promiscuity in the ligandspecificity of GFRa-1 and GFRa-2, the physiologicalrelevance of these findings has yet to be ascertained.

An additional member of the GFRa family, GFRa-3,has recently been identified by searching mouse andhuman EST databases for sequences related to GFRa-1and GFRa-2 (Jing et al., 1997; Naveilhan et al., 1998). LikeGFRa-1 and GFRa-2, GFRa-3 has the C-terminal fea-tures of GPI-linked proteins, although its ligand is notknown. GFRa-1, GFRa-2, and GFRa-3 are widely ex-pressed in the central nervous system and peripheralnervous system and in a variety of other tissues andorgans during development (Durbec et al., 1996; Jing etal., 1996; Treanor et al., 1996; Trupp et al., 1996; Vega et al.,1996; Worby et al., 1996; Baloh et al., 1997; Buj-Bello et al.,1997; Creedon et al., 1997; Jing et al., 1997; Klein et al.,1997; Molliver et al., 1997; Sanicola et al., 1997; Suvanto etal., 1997; Widenfalk et al., 1997; Naveilhan et al., 1998).However, whereas the expression of GFRa-1 and GFRa-2is maintained in the adult, GFRa-3 expression is barelydetectable in adult tissues and organs (Naveilhan et al.,1998). Like members of the GFRa family, Ret is alsowidely expressed in the nervous system and otherorgans and tissues (Pachnis et al., 1993; Schuchardt et al.,1995; Tsuzuki et al., 1995).

RESULTS AND DISCUSSION

Cloning and Structural Features of GFR a-4

A cDNA clone encoding GFRa-4, a novel member ofthe GFRa family of receptors, was isolated by screening

an E10 chicken brain cDNA library with a mouseGFRa-1 probe (gift of Arnon Rosenthal). A second,independent GFRa-4 cDNA clone was obtained byscreening an E6 chicken brain cDNA library with aprobe derived from the original GFRa-4 cDNA. Thenucleotide sequence of chicken GFRa-4 predicts a 431-amino-acid protein that is more closely related to GFRa-1and GFRa-2 than to GFRa-3. Sequence alignment (Fig.1) showed that whereas GFRa-4 has approximately 40%amino acid identity with both mouse and chickenGFRa-1 and GFRa-2, it has only 27% identity withmouse GFRa-3. This compares with 48% identity be-tween chicken GFRa-1 and GFRa-2 (Buj-Bello et al.,1997). Of the 30 cysteines that are conserved betweenGFRa-1 and GFRa-2, 28 are conserved in GFRa-4(Fig. 1). Like other members of the GFRa family, GFRa-4possesses an N-terminal, hydrophobic, putative signalpeptide for secretion and the characteristic features ofGPI-linked proteins, namely, a C-terminal hydrophobicdomain separated by a hydrophilic linker region from acleavage/binding consensus sequence for GPI linkage(Gerber et al., 1992).

Expression of GFR a-4 in the Chicken Embryo

We used Northern blotting, semiquantitative RT/PCR, and in situ hybridization to analyze the expressionof GFRa-4 mRNA in the chicken embryo during devel-opment. Northern blotting revealed a transcript ofapproximately 3.0 kb in the kidney, skeletal muscle,skin, intestine, and lung of E10 embryos. An additional,minor 3.7-kb transcript was evident in kidney, the tissuewith the highest levels of expression by far. GFRa-4mRNA was barely detectable by Northern blotting inthe liver and heart of E10 embryos (Fig. 2). In contrast,Northern analysis of GFRa-1 and GFRa-2 mRNA expres-sion at this age in the chicken embryo revealed highlevels in the intestine and liver, respectively (Buj-Bello etal., 1997). Within the CNS, the spinal cord expressed thehighest level of GFRa-4 mRNA; the medulla oblongata,pons, cerebellum, and midbrain expressed lower levels,and within the forebrain only very low levels weredetectable (Fig. 2). The level of GFRa-1 mRNA was alsohigher in the spinal cord than in the brain of the E10chicken embryo, although within the brain the forebrainexpressed the highest levels of GFRa-1 mRNA at thisstage (Buj-Bello et al., 1997). In contrast, GFRa-2 mRNAwas expressed at uniform levels in all regions of thechicken CNS at E10 (Buj-Bello et al., 1997).

Comparison of the relative levels of GFRa-4 mRNA ina variety of tissues and organs at E18 by semiquantita-tive RT/PCR revealed high levels in the kidney and

118 Thompson et al.

FIG. 1. Aligned amino acid sequences of mouse GFRa-1, GFRa-2, and GFRa-3 and chicken GFRa-1, GFRa-2, and GFRa-4. Conserved cysteinesare shown in red, and amino acids in GFRa-1, GFRa-2, and GFRa-3 that are identical to those in GFRa-4 are shown in blue. The N-terminal,hydrophobic, putative signal peptides are underlined in green, the C-terminal hydrophobic domain is underlined in red, and the putativebinding/cleavage consensus sequences for GPI linkage are enclosed in red boxes. GenBank accession numbers: mouse GFRa-1, 2494709; chickenGFRa-1, U90541; mouse GFRa-2, 2494713; chicken GFRa-2, U90542; mouse GFRa-3, 2674177; chicken GFRa-4, AF045162.

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brain, lower levels in skin, and very low levels in liver,heart, lung, muscle, stomach, and intestine (Fig. 3). Incontrast, whereas the level of GFRa-2 mRNA was highin the liver, lower in the brain and skin, and very low inother tissues and organs, GFRa-1 mRNA was expressedat a similar levels in most tissues and organs except theheart and muscle where the level was very much lower(Fig. 3). Ret mRNA was expressed at higher levels inbrain, liver, and intestine than in other tissues (Fig. 3).

Semiquantitative RT/PCR was also used to study thedevelopmental changes in the expression of GFRa-1,GFRa-2, GFRa-4, and ret mRNAs relative to L27 mRNA(which encodes a ubiquitous, constitutively expressedribosomal protein). In some tissues, there were similardevelopmental changes in the expression of GFRa-1,GFRa-2, GFRa-4, and ret mRNAs. For example, be-tween E6 and E18 the levels of these mRNAs increasedin brain and decreased in muscle and intestine (Fig. 4).In some tissues, the relative levels of some of thesemRNAs, such as GFRa-1 and GFRa-4 mRNAs in thekidney, remained fairly constant. In other tissues, thesemRNAs displayed different temporal patterns of expres-sion. For example, whereas the level of GFRa-1 mRNAin heart gradually decreased between E6 and E14,GFRa-2, GFRa-4, and ret mRNAs exhibited a clearincrease over this period of development.

In situ hybridization showed that GFRa-4 mRNA isprominently expressed in several neuronal populationsin the developing CNS (Fig. 5). These include neurons ofboth the dorsal and the ventral gray columns of thespinal cord, the Purkinje cells, granule cells, and neu-rons of the deep nuclei of the cerebellum. Althoughmotoneurons of the ventral gray column are moreprominently stained than cells of the dorsal gray col-

umn, this difference may reflect the larger perikarya ofmotoneurons rather than differences in expression level.The pattern of hybridization in the intestine is consistentwith expression of GFRa-4 mRNA in the myentericplexus and mucosal epithelium (Fig. 5). Prominentexpression was also observed by in situ hybridization inthe proventricular glands of the stomach and in thedeveloping kidney. No staining was observed in sec-tions hybridized with a sense control probe.

Taken together, the above studies demonstrate thatGFRa-1, GFRa-2, GFRa-4, and ret mRNA have overlap-ping, though distinct, patterns of expression in thechicken embryo during development. Although, likeGFRa-3, the ligand for GFRa-4 has yet to be identified,the distinctive pattern of GFRa-4 expression indicatesthat GFRa-4 has distinctive developmental and physi-ological roles.

FIG. 3. Autoradiograms showing the RT/PCR products for GFRa-1,GFRa-2, GFRa-4, and ret in a variety of tissues at E18 and in lung,skeletal muscle, and intestine at E14, E6, and E10, respectively, theages at which GFRa-4 mRNA expression was highest in these tissues.The same mRNA preparations were used for all reactions with each setof primers and were diluted appropriately to contain very similarlevels of L27 mRNA, as shown by the similar levels of the RT/PCRproducts for L27 mRNA in the bottom autoradiogram. Controlreactions from which the reverse transcriptase step was omitted(controls 1 and 2 which contain extracted RNA from 6 of the 12 tissues)show that there was no detectable contamination by genomic DNA.Very similar results were obtained from a separate set of tissues.

FIG. 2. Analysis of the expression of GFRa-4 mRNA by Northernblotting. Similar amounts of total RNA from various embryonictissues and CNS regions were transferred to filters and hybridizedwith the GFRa-4 probe. The 3.0-kb GFRa-4 transcript is indicated.

120 Thompson et al.

FIG. 4. Autoradiograms showing the RT/PCR products for GFRa-1, GFRa-2, GFRa-4, and ret at E6, E10, E14, and E18 in heart, lung, and skeletalmuscle (A), skin, brain, and stomach (B), and liver, kidney, and intestine (C). The same mRNA preparations were used for all reactions with eachset of primers and were diluted appropriately to contain very similar levels of L27 mRNA, as shown by the similar levels of the RT/PCR productsfor L27 mRNA in the bottom autoradiograms in the age set for each tissue. Control reactions from which the reverse transcriptase step was omitted(not shown) were carried out for each reaction and were negative in all cases, demonstrating that there was no detectable contamination bygenomic DNA in any of the preparations. The autoradiograms were developed for different lengths of time so that very weak bands could beobserved. Very similar results were obtained from a separate set of tissues.

GFRa-4, a New GDNF Family Receptor 121

122 Thompson et al.

EXPERIMENTAL METHODS

GFRa-4 Cloning

An E10 chicken brain cDNA library in lambdaZAP II(Stratagene) was screened using a 32P-labeled, nick-translated probe generated from the full coding regionof mouse GFRa-1 (gift of Arnon Rosenthal) at lowstringency (hybridization at 59°C, final wash at 65°C in23 SSC, 0.2% SDS) as described previously (Baka et al.,1996). In addition to several clones encoding GFRa-1and GFRa-2 (Buj-Bello et al., 1997), we isolated a cloneencoding the 38 coding sequence of a novel, related,putative receptor that we have termed GFRa-4. Al-though we were not successful in obtaining full-lengthGFRa-4 cDNA clones by rescreening the E10 braincDNA library, a clone encoding the full coding sequenceof GFRa-4 was obtained by screening an E6 chickenbrain cDNA library (Stratagene) at high stringency(hybridization at 60°C, final wash at 65°C in 0.23 SSC,0.2% SDS) with a 32P-labeled, nick-translated probegenerated from the first GFRa-4 cDNA.

Northern Blot Hybridization

Northern blotting (Baka et al., 1996) was used tomeasure the levels of GFRa-4 mRNA in various tissues.Total RNA was run on formaldehyde gels and blottedonto Hybond filters which were hybridized with a32P-labeled, nick-translated probe comprising a unique800-bp fragment of the GFRa-4 sequence generated byPCR using the primers 58-CAGAGTTCCAGTTTAAT-TGC-38 and 58-CCCGAAAGAAGTTCCTTGTC-38. Thefilters were prehybridized at 42°C for 4 h and werehybridized with the probe at 42°C for 24 h. The filterswere washed three times with 23 SSC and three timeswith 23 SSC plus 0.2% SDS before exposure to FujiX-ray film at 270°C with intensifying screens.

Detection of GFR a-1, GFRa-2, GFRa-4,and ret mRNAs by RT-PCR

A semiquantitative RT/PCR assay was used to deter-mine the expression pattern of GFRa-1, GFRa-2, GFRa-4,

and ret mRNAs in various embryonic tissues. TotalRNA was extracted (Chomczynski and Sacchi, 1987)from brain, heart, lungs, stomach, intestine, liver, kid-neys, skin, and muscle of E6, E10, E14, and E18 chickenembryos. Following DNase treatment, RNA was furtherpurified using an RNAID kit (BIO 101) and was recov-ered in 50–400 µl (depending on the tissue size) ofDEPC-treated water. Quantitative, competitive RT/PCRwas used to determine the amount of mRNA for thehousekeeping L27 ribosomal protein in each RNA sam-ple (Allsopp et al., 1993), thus allowing total RNAsamples from E6 to E18 to be appropriately dilutedso that they contained the same concentration of L27mRNA. Two microliters of total RNA was reversetranscribed for 45 min at 37°C with Gibco BRL Super-script enzyme in a 50-µl reaction containing themanufacturers’ buffer supplemented with 0.5 mMdNTPs and 10 µM random hexanucleotides. A 5-µlaliquot of each reverse transcription reaction was thenamplified in a 50-µl PCR containing 13 PC2 buffer(Helena BioSciences), 0.1 mM dNTPs, 2 units of TaqSupreme (Helena BioSciences), and 40 ng of 32P 58end-labeled primers. The forward primers for eachcDNA were 58-ACCTGAGAAGGAGGATGG-38 (GFRa-1), 58-CCTTTGTGGATCAGAAGGC-38 (GFRa-2), 58-TGCGAAGACACAGCCTGTGCT-38 (GFRa-4), and 58-ATCTGTGCCAGAAGTCTC-38 (ret). The reverse primersfor each cDNA were 58-TGACATCCTTGATAATCT-38(GFRa-1), 58-AGCTTCAGCAGCACAATGG-38 (GFRa-2), 58-GCATAACGCGACCTACAGACG-38 (GFRa-4),and 58-AGTCTTCTCTATCTAGGC-38 (ret). The ampli-fied products for GFRa-1, GFRa-2, GFRa-4, and ret were97, 82, 121, and 163 bp, respectively. GFRa-1 andGFRa-2 cDNAs were amplified by cycling for 1 min at95°C, 1 min at 50°C, and 1 min at 68°C. The cyclingconditions were the same for GFRa-4, ret, and L27,except for the annealing temperatures which were 60,53, and 58°C, respectively. To ascertain the reproducibil-ity of the results, each PCR was repeated twice for twodifferent numbers of cycles. These were 22–24 cycles forGFRa-1, 21–23 cycles for GFRa-2, 25–27 cycles forGFRa-3, 25–27 cycles for ret, and 19 cycles for L27. Thecycling conditions were optimal for the amplification of

FIG. 5. Localization of GFRa-4 mRNA in tissue sections of E18 embryos by in situ hybridization. (A) Section through the wall of theproventiculus of the stomach showing prominent labeling of the proventricular glands (p), mucosa (m), and ganglia of the myenteric plexus(arrow). The lumen of the proventriculus (l) lies to the right. (B) Transverse section through the lumbar spinal cord showing labeled neuronsthroughout the gray matter, although labeling is more intense among the motoneurons of the anterior gray column (arrows). (C) Section throughthe intestine showing prominent labeling in the mucosa and ganglia of the myenteric plexus (arrow). (E) Section through the kidney showinglabeling in the developing renal tubules and glomeruli. (D and F) Sections through the cerebellum showing very prominent labeling of Purkinjiecells (arrows) and neurons of the deep cerebellar nuclei (d). Granule cells in the external granular layer (e) and internal granular layer (i) are alsoprominently labeled. Bars, 200 µm (the bar in E applies to A–E).

GFRa-4, a New GDNF Family Receptor 123

each transcript so that the rate of reaction does notplateau. The reaction was then completed with a 10-minextension at 68°C. The RT/PCR products were run on8% nondenaturing polyacrylamide gels that were subse-quently dried and autoradiographed.

In Situ Hybridization

In situ hybridization was performed on 25-µm cryo-stat sections of E18 chicken embryos. Hybridization wascarried out with cRNA probes made by run-off transcrip-tion from a linearized pGEM-T vector containing a620-bp fragment of GFRa-4 cDNA generated by the PCRprimers 58-GAGGTGACCCAGGTGACCCGC-38 and 58-GCGTTCAGGTAGGTCCCGTTCG-38. Sense and anti-sense RNA transcripts (made by using Sp6 and T7promoters, respectively) were labeled by digoxygenin(Boehringer Mannheim) and hybridized at 55°C withthe sections as described previously (Baka, 1996). Thiswas followed by washing in 0.13 SSC at 55°C anddetection of the hybridized probe with the BoehringerMannheim detection kit.

ACKNOWLEDGMENTS

Our thanks are extended to Arnon Rosenthal for the mouse GFRa-1cDNA and to David Roche and Jim Allan for preparing the illustra-tions. This work was supported by grants from the Wellcome Trustand Medical Research Council.

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Received February 3, 1998Revised March 19, 1998

Accepted March 19, 1998

126 Thompson et al.