From the Ludwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
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ABSTRACT |
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We report the genomic organization of the mouse
orphan receptor related to tyrosine
kinases (Ryk), a structurally unclassified member of the growth factor receptor family. The mouse RYK protein is
encoded by 15 exons distributed over a minimum of 81 kilobases. Genomic
DNA sequences encoding a variant protein tyrosine kinase ATP-binding
motif characteristic of RYK are unexpectedly found in two separate
exons. A feature of the gene is an unmethylated CpG island spanning
exon 1 and flanking sequences, including a TATA box-containing putative
promoter and single transcription start site. Immunohistochemical
examination of RYK protein distribution revealed widespread but
developmentally regulated expression, which was spatially
restricted within particular adult organs. Quantitative
reduction of Southern blotting stringency for the detection of
Ryk-related sequences provided evidence for a
retroprocessed mouse pseudogene and a more distantly related gene
paralogue. Extensive cross-species reactivity of a mouse
Ryk kinase subdomain probe and the cloning of a
Ryk orthologue from Caenorhabditis elegans
demonstrate that Ryk and its relatives encode widely
conserved members of a novel receptor tyrosine kinase subfamily.
Members of the receptor tyrosine kinase
(RTK)1 family of
transmembrane signal transduction proteins participate in the
regulation of diverse cellular activities such as mitogenesis,
motility, differentiation, survival, metabolism, adhesion,
fasciculation, morphogenesis, and oncogenesis (1). Structurally, and
usually functionally, related RTKs are grouped into distinct
subfamilies largely on the basis of characteristic combinations of
protein motifs in their ligand-binding extracellular domains. Unique in this classification system is the RYK (for related to
tyrosine kinase) receptor subfamily which,
aside from two leucine-rich motifs in the mammalian orthologues and a
possible protease cleavage site common to all RYKs, is comprised of
members devoid of recognizable protein modules in their extracellular
domains characteristic of other RTKs (2-4).
The cytoplasmic protein tyrosine kinase (PTK) activity of RTKs is
classically activated by homodimerization of receptor protomers mediated by the binding of an extracellular growth factor ligand (5).
More recently, RTK activation via heterodimerization (6, 7),
extracellular matrix (8), and ligand-independent routes (9) have been
recognized as physiologically significant modes of signal generation.
Twelve conserved peptide sequence motifs, or subdomains, are the
signature of the PTK catalytic domain, including some 13 invariant
residues, which participate in the phosphotransfer reaction (10,
11).
A subclass of RTKs that lack demonstrable phosphotransferase activity
has emerged recently whose members display substitutions in one or more
of the conserved and catalytically important PTK motifs. These include
the human CCK4 (12) and chicken KLG (13) orthologues, ERBB3 (14), the
human and mouse Eph-like proteins HEP (15) and MEP (16), and the human
RYK protein (3). The likely functional importance of RYK
subfamily-specific amino acid substitutions to otherwise universally
conserved PTK motifs is underscored by their occurrence in such
phylogenetically diverse species as Drosophila
(Doughnut2 and
Derailed, Ref. 4), human (3), mouse (2) and zebrafish Ryk.3
Although largely functionally uncharacterized, transmembrane proteins
such as RYK which bear an apparently catalytically inactive PTK domain
may well modulate or relay growth-regulatory cues present in the
extracellular environment to cytoplasmic signaling and/or effector
molecules. For instance, inactivation of the PTK activity of the
Caenorhabditis elegans Eph receptor VAB-1 results in weak mutant phenotypes (relative to extracellular domain mutations), suggesting that a subset of RTKs may execute kinase-independent functions (17). These may include recruitment of heterologous protein
kinase-competent receptor subunits, as in the case of a
kinase-defective EGFR mutant still capable of activating
mitogen-activated protein kinase (18). That mice homozygous for a
deletion of exons encoding the PTK domain of VEGFR1 (Flt-1), an RTK
essential for embryonic angiogenesis, exhibit normal vessel development and are fully viable suggests that the primary biological role of this
receptor is in ligand binding rather than signal transduction (19).
Identification of derailed in a P element enhancer trap
screen for fasciculating neurons demonstrated a requirement for growth cone pathfinding cues transduced by this RYK subfamily member during
Drosophila nervous system development (4). Furthermore, defective somatic muscle insertion site selection into the epidermis of
derailed mutants suggests that the mechanism for muscle
insertion target recognition is biochemically similar to that used in
axon pathfinding (20). A pleiotropic role for ERBB3 in cardiac and central nervous system development has been demonstrated in
ErbB3-null mice (21, 22). However, a role for other
PTK-inactive RTKs in growth regulation remains to be demonstrated.
Here we report the structure of the mouse Ryk transcription
unit, including the likely location of its promoter, as identified by
an unmethylated CpG island. The unusual organization of domains in the
mouse RYK protein extends to the genomic structure that displays
features unique within the RTK/growth factor receptor gene family.
Southern blotting results suggestive of the existence of a
retroprocessed mouse Ryk pseudogene are also presented.
Immunohistochemical analysis has been undertaken to define the spatial
distribution of RYK expression within individual adult mouse organs.
Conserved structural elements that define the RYK subfamily are
identified in five metazoan proteins, including one from C. elegans, indicating an early evolutionary origin of these RTK-like
molecules. Consistent with subsequent duplications of the ancestral
vertebrate genome, the mouse, human, and zebrafish genomes also contain
putative Ryk subfamily paralogues that we find to be
phylogenetically conserved molecules with likely roles in cellular
growth regulation.
Isolation and Characterization of Genomic Clones--
Lambda
phage from a mouse 129/Sv genomic DNA library (Stratagene, La Jolla,
CA) were screened with probes generated from the mouse Ryk
cDNA (2) by PCR. The extracellular domain probe was amplified using
the primers 5'-TCGGCTTCCGCGGGGCCCAGC-3' (human cDNA nt 201-222)
and 5'-ACACACGCGTGGAAGTCGTTGGC-3' (mouse cDNA nt 737-715), and the
kinase domain probe was amplified using the primers
5'-TTGGGCGTATTTTCCATGGGA-3' (mouse cDNA nt 1083-1103) and
5'-TGGACCAGCTGCTGGAACTTA-3' (mouse cDNA nt 1842-1822). Hybridizing phage were plaque-purified, and phage DNA was prepared as described (23). Phage DNA inserts were excised with NotI, subcloned
into plasmid vectors, and restriction mapped by partial digestion with EcoRI, HindIII, and XbaI. A mouse
129/SvJ genomic DNA library (release I) in BACs was screened by Genome
Systems, Inc. (St. Louis) by PCR using oligonucleotides that generate a
221-bp product from the 5' end of genomic subclone pBS Ryk
9.2.2. (BAC.01, 5'-GTCGCATCCCTTCCTTCCACTTTA-3' and BAC.02,
5'-GCCCAGACCCACATGCCCAGAG-3'). BAC DNA was prepared using a modified
Qiagen plasmid maxi kit protocol.
Southern Blotting--
Twenty micrograms of mouse 129/Sv genomic
DNA was digested to completion with the indicated restriction
enzyme(s), fractionated on 0.8% agarose gels, and transferred to
GeneScreen Plus® membrane (NEN Life Science Products) as described
(24). Membranes were prehybridized in 10% dextran sulfate, 1 M NaCl, 1% SDS, 100 µg/ml sheared and denatured salmon
sperm DNA at 65 °C for >30 min. Random-primed DNA probes were
labeled with [
Reduced stringency Southern blotting for characterization of
Ryk-related genes was performed as described (25). The 1×
hybridization solution was modified to 2.5× Denhardt's solution, 10%
dextran sulfate, 10 mM Tris-HCl, pH 7.5, 5× SSC, 1% SDS,
100 µg/ml sheared and denatured salmon sperm DNA and 20-50% (v/v)
formamide. Probes used were produced by PCR from the mouse
Ryk cDNA (2) using primers specific for the
extracellular domain, 5'-TCTTGATGCAGAAGCTTTACT-3' (nt 293-305) and
5'-GAAATATCACTGCACAGC-3' (nt 776-759) or the kinase domain,
5'-AAACCTCCTTCCTATTACTC-3' (nt 1234-1253) and
5'-CACGTCACTAGCACTAGAG-3' (nt 1635-1617). Cross-species Southern
("Zoo") blotting was performed using 15 µg of genomic DNA
isolated from each species (with the exception of
Drosophila, 10 µg, and E. coli, 2 µg) and
digested to completion with HindIII. A 159-bp mouse
Ryk exon 13 probe (encoding the PTK motifs VII, VIII, a
portion of IX and the putative activation loop) was synthesized by PCR
using the cDNA as template and the primers 5'-CATCGACGACACTCTTCA-3'
(nt 1477-1494) and 5'-CACGTCACTAGCACTAGAG-3' (nt 1635-1617).
Hybridization and washing conditions employed 1× reduced stringency
hybridization solution containing 20% formamide ( DNA Sequencing--
Double-stranded plasmid DNA was sequenced by
the dideoxy chain termination method (PRISMTM Ready
Reaction DyeDeoxyTM Terminator Cycle Sequencing kit;
Perkin-Elmer). A 1.8-kb XbaI-BglI fragment
flanking the presumptive initiator methionine was sequenced on both
strands by generating a set of nested 200-300-bp deletions in both
directions using an Exo-Size Deletion Kit (New England Biolabs,
Beverly, MA; Ref. 26).
Cloning of Ceryk--
A search of a C. elegans
database (ACeDB) using the Doughnut extracellular domain revealed
homology with an open reading frame on genomic cosmid C16B8 designated
C16B8.1 (27), here referred to as Ceryk. To verify the
exon-intron structure predicted by the Genefinder program (28),
oligonucleotides to putative Ceryk exons were used to
amplify the entire coding sequence, and flanking regions, in
overlapping RT-PCR products. Reverse transcription of mixed stage
C. elegans total RNA (~5 µg) was performed using a first
strand synthesis for RT-PCR kit (Amersham); cDNA amplification by
touchdown PCR was performed as described (29). Detection of sorting
signals and cleavage sites in the N termini of the RYK proteins was
performed using SignalP, and transmembrane domains were identified
using PSORT.
PCR--
Amplification of human RYK exons from
genomic DNA (~100 ng) was performed as described above for PCR of
C. elegans cDNA. Oligonucleotides were designed to
amplify a region of the human RYK gene homologous to that
contained in the mouse Ryk Zoo blot probe,
5'-CATTGATGACACACTTCAAG-3' (human cDNA nt 1539-1558) and
5'-CACATCACTAGCGCTAGAG-3' (human cDNA nt 1698-1679), which amplify
a 159-bp product from human cDNA. The oligonucleotide probe
5'-CTTTCAAGAGCCATCCAAC-3' (human cDNA nt 1661-1643) is fully
internal to the primers used in the PCR.
Sequence Analysis of the Ryk Promoter--
Identification of
putative transcription factor binding elements was performed by
searching position-weighted nucleotide distribution matrices using
MatInspector (30). The CpG island was analyzed with the EGCG program
CpGplot using the algorithms of Gardiner-Garden and Frommer (31).
Production of Monoclonal Antibodies against the Human RYK
Extracellular Domain--
A purified FLAG-tagged version of the human
RYK extracellular domain (RYK-EX-FLAG) was used to immunize female
BALB/c mice prior to fusion of spleen cells with the mouse myeloma
P3X63Ag.653 (NS-1). Hybridoma supernatants containing monoclonal
antibodies to the RYK extracellular domain were screened for by an
enzyme immunoassay. Antibodies chosen for further analysis (RYK-1,
RYK-2, and RYK-3) were subcloned twice and isotyped as described
(32).
Immunohistochemistry--
Tissue sections were stained with a
two-step immunoperoxidase technique as described (33), with the
modification that the RYK-1 monoclonal was detected with a goat
anti-mouse IgM-horseradish peroxidase secondary reagent (Dako,
Copenhagen, Denmark).
Structure of the Mouse Ryk Gene--
The mouse RYK protein is
encoded by 15 exons covering a minimum of 81 kb (Fig.
1a). Twenty four independent
phage clones isolated from a 129/Sv genomic lambda library and two
bacterial artificial chromosome (BAC) clones identified by
amplification with the primer pair BAC.01 and BAC.02 (Fig.
1c) were analyzed to define the organization of mouse
Ryk. A 4-kb XbaI fragment containing exon 1, a
CpG island, the transcription start site, and putative promoter
sequences (Fig. 1, a and b) was subcloned from
BAC.Ryk.2. The exact size of intron 1 (>19 kb) has not been
determined.
Two possible translation initiation codons (M1 and M2; see Fig.
2b) are located in exon 1 which has a G + C content of 83%. No in-frame ATG codons are present
upstream of M1 before the next in-frame stop codon. Exons 1-5 encode
the RYK extracellular domain (exons 2 and 4 each encode a putative
leucine-rich motif, LRM), exon 6 the hydrophobic transmembrane domain,
exons 7 and 8 the serine-threonine-rich juxtamembrane domain, and exons
8-15 the PTK-like domain. A 15-residue C-terminal tail is also encoded by exon 15. That the signal peptide, two LRMs, and single transmembrane domain are each encoded by an individual exon suggests that the Ryk gene was originally assembled from functional units by
exon shuffling. A variant of the ATP-binding motif (subdomain I,
consensus GXGXXG; Ref. 11), recognizable as
QEGTFG in mouse RYK, is encoded by exons 8 and 9. In all mammalian RTKs
reported to date, this motif is encoded by a single exon, for example
the human INSR (34), c-MET (35), and
c-KIT (36) genes. The PTK motif IX, responsible for
stabilization of the catalytic loop (11), is also encoded by two exons
of the mouse Ryk gene (exons 13 and 14).
Six independent groups have now cloned and sequenced the mouse
Ryk cDNA (2, 37-41). When these sequences are aligned
(not shown), nucleotide differences that predict amino acid
substitutions are apparent. The major differences are found in the 5'
region of the cDNA and most likely reflect difficulties in
sequencing this highly G + C-rich region. No nucleotide ambiguities
were encountered in our genomic sequencing of this portion of the open reading frame, the sequence of which always matched a majority of
published cDNAs, consistent with the interpretation that
differences between the reported sequences are the result of sequencing
errors. The long 5'-untranslated region published by Kelman et
al. (39) appears to be part of a chimeric cDNA clone given
that the sequence is unrelated to genomic DNA sequence in Fig.
2b beginning at position 1763 and extending upstream.
Additionally, our BAC.Ryk clones do not hybridize with
oligonucleotides complementary to the unique 5'-untranslated end
reported by Kelman et al. (39). The kinase activation loop
of mouse RYK, bounded by subdomains VII and VIII of the PTK catalytic
core (10), is encoded by exon 13 and contains a conserved tyrosine
residue at position 454 (Ref. 2; see Fig. 6) which is often involved in
catalytic autoregulation of diverse PTK superfamily members (42). The
two alternative polyadenylation signals reported by Kelman et
al. (39), both of which perfectly match the consensus sequence
AATAAA, reside in exon 15 (Fig. 1a).
The sequences of mouse Ryk exon-intron boundaries are
presented in Table I. The mouse
Ryk transcription start site (see below) defines the 5'
boundary of exon 1. All introns conform to the 5'-gt ... ag-3'
motif, with the exception of exon 8 which has a modified 3' splice
acceptor site (Table I).
Identification of a CpG Island Containing the Putative Mouse Ryk
Promoter--
The DNA sequence flanking exon 1 is shown in Fig.
2b. No consensus sequence for a splice acceptor site
associated with this exon was found in the upstream region. This and
the location of the transcription start site (see Discussion) suggest
that the promoter resides at least partly within this sequence. The
presence of a CpG island in this area, recognizable as a high density
of sites for restriction enzymes with G + C-rich recognition sequences containing one or more CpG dinucleotides (Fig. 2a; Ref. 43), is a landmark for the Ryk promoter since a CpG island was
always found to cover the whole or part of the promoter in a survey of 102-island-containing genes expressed in a wide variety of tissues (44).
CpG islands are defined as sequences >200 bp in size comprising a
moving average G + C content >50% and an observed/expected CpG
dinucleotide frequency >0.6 (31, 44). The 859-bp Ryk CpG island lies between nucleotides 1469 and 2347 and is characterized by
elevated G + C content (75%), an observed/expected CpG ratio of 1.004 (Fig. 3a), and is unmethylated
at CpG residues (Fig. 3b). Mouse genomic DNA prepared from
low passage embryonic stem cells (W9.5 line, derived from mouse 129/Sv
C3
Sequences immediately flanking the transcription start site (see
"Discussion") display potential binding motifs for a variety of
transcriptional modulators (Fig. 2b). The MatInspector
program (30) was used to search the sequence for high quality matches to a data base of position-weighted nucleotide distribution matrices. DNA elements potentially mediating the response to growth-regulatory stimuli through the sequence-specific binding of transcription factors
are particularly abundant in the putative Ryk promoter (Fig.
2b). These include response elements for transcription
factors activated by mitogenic (e.g. Ets family, AP-1,
n-Myc, Egr family, E2F, RREB-1, NGFI-C, c-Myb), anti-mitogenic (IRF-1,
ISRE), metabotropic (T3 receptor, aryl hydrocarbon
receptor, and nuclear translocator), and differentiation (HNF-3 Identification of Mouse Ryk-related Sequences by Southern
Blotting--
A screen of the mouse genome by Southern blotting using
mouse Ryk cDNA-derived probes was initiated as a first
step in resolving the issue of whether mammals have one or more
Ryk gene paralogues (see Ref. 41). Restriction fragment
length polymorphism mapping in the mouse (50) and human cytogenetic
analysis (3) have previously identified loci on chromosomes 12 and
17p13.3, respectively, with high homology to the canonical mammalian
Ryk cDNAs. Consistent with this, most RTKs belong
to subfamilies of homologous receptors that engage in functional
interactions (e.g. the ErbB subfamily, Refs. 6 and 51; the
platelet-derived growth factor receptor, FGFR, and Trk subfamilies,
Ref. 7; the INSR subfamily, Ref. 52).
By using a gradient of hybridization and washing stringencies
originally employed to detect novel human RTKs by Southern blotting of
genomic DNA (25, 53), we have detected two classes of mouse genomic DNA
fragments related to the Ryk gene characterized herein. The
first class is detectable at high stringency (Fig.
4c, arrows) using
an Ryk kinase domain probe. However, only the hybridization signals expected from the structure of the mouse Ryk gene
(Fig. 1a) are observed when genomic DNA digests are
hybridized at high stringency with a probe to the Ryk
extracellular domain (Fig. 4a). The nature of this first
class of Ryk-related hybridization signals (high signal
strength of the fragments, in particular the 3.8-kb EcoRI
and 2.2-kb XbaI fragments, at high stringency) is consistent
with a contiguous organization of kinase domain exons comprising a
partially retroprocessed pseudogene ( Immunohistochemical Analysis of RYK Protein Expression in the
Mouse--
Analysis of Ryk mRNA expression in a large
sample of mouse and human tissues has been reported by us and others
(2, 37-41, 54-57). These analyses show that Ryk mRNA
is distributed ubiquitously. In order to assess the spatial
distribution of the functional gene product, we generated an anti-RYK
monoclonal antibody and performed immunohistochemistry on sections of
embryonic and adult mouse tissues. The RYK-1 antibody was raised
against the extracellular domain of human RYK (Fig.
5a) and is specific for the
extracellular domain of this receptor (Fig. 5, b and
c). The RYK-1 monoclonal antibody has been used in Western
blotting analysis of cell lines overexpressing a variety of RTK
extracellular domains (e.g. EGFR, VEGFR1, VEGFR2, VEGFR3,
and Tie2) with no evidence of cross-immunoreactivity. The dual Western
blotting signal from MCF-7 lysate at approximately 90 and 47 kDa (Fig.
5c) is also observed in reduced immunoprecipitates of
metabolically labeled MCF-7 cells using an antiserum raised against the
C terminus of human RYK (3) and is consistent with proteolytic
processing of the receptor extracellular domain at the tetrabasic KRRK
site. Disulfide bonding between Cys-156 and Cys-191 of the human
receptor, which represent conserved residues that flank the KRRK motif
in all RYK subfamily members (see Fig. 6), may be an important
post-translational modification. The RYK-1 antibody cross-reacts with
both mouse and zebrafish RYK but not with Drosophila
Derailed.4
RYK protein expression was detected in a large proportion of adult
mouse tissues and on cells from a variety of origins. In the kidney,
strongest RYK staining is seen in tubules immediately under the cortex
(Fig. 5d), which gradually declines in strength toward the
pelvic region. Glomeruli within all regions of the kidney are uniformly
unstained with the RYK-1 antibody. RYK expression is limited to
epithelial cells lining the renal tubules. Strong and uniform RYK
expression is evident in liver lobules, associated with the membranes
of hepatocytes (Fig. 5f). The adrenal medulla shows no
reactivity with the RYK-1 antibody, whereas high RYK expression is seen
in the adrenal cortex (Fig. 5h). Strong and uniform RYK
expression is present on embryonic day 14 myocardium (Fig.
5j); this represents the earliest expression of RYK that we
have observed in the mouse (embryonic days 8-12 are unstained; not
shown). Adult spleen shows staining with the RYK-1 antibody restricted
to the red pulp (Fig. 5l). Within this compartment, megakaryocytes show membrane-localized immunoreactivity, whereas erythroblasts and lymphocytes are free of staining. In addition, reticular fibers and endothelial cells lining the venous sinuses are
stained. The adult small intestine displays strong staining with the
RYK-1 antibody on the epithelium and connective tissue/vascular core of
villi, whereas the glandular crypts are unstained (Fig. 5n).
The mucosa of the large intestine is stained on connective tissue
surrounding intestinal glands, which are themselves free of RYK-1
immunoreactivity (Fig. 5p).
Phylogenetic Conservation of the Ryk Gene
Subfamily--
Complementary DNAs encoding RYK subfamily members have
been isolated from mouse (2, 37-41), human (3, 54, 57), and zebrafish,
and two orthologues, doughnut and derailed (4), have been cloned from Drosophila. We report here the
cloning, by RT-PCR, of a C. elegans ryk orthologue
(Ceryk) that bears the characteristic features of this RTK
subfamily (Fig. 6). The cDNA predicts a 583-residue pre-protein
containing an N-terminal hydrophobic signal sequence and a
transmembrane domain.
To explore the range of evolutionary conservation of Ryk
subfamily members, low stringency Southern blot hybridization of a
mouse exon 13 probe to a "Zoo blot" was performed. Extensive and
strong hybridization of the Ryk-specific kinase subdomain probe to genomic DNA from a wide range of metazoans was observed (Fig.
7a). The 3.6-kb mouse
HindIII fragment represents the expected hybridization
signal from mouse Ryk, and the strong ~0.7-kb signal, which is also seen at high stringency in Fig. 4c, is most
likely derived from a mouse pseudogene. Weaker hybridization to other mouse genomic HindIII species is also visible (at ~1.3,
~1.5, and ~1.8 kb, see also Fig. 4d). To identify the
origins of the two strongest hybridization signals from human genomic
DNA (Fig. 7a), PCR was employed to map the organization of
human RYK sequences recognized by the Zoo blot probe. Using
primers to human RYK corresponding to those used to produce
the mouse exon 13 Zoo blot probe, a 159-bp PCR product, which was
uninterrupted by introns and not cleavable by HindIII (3),
was amplified from genomic DNA (Fig. 7b). This result
indicates that the mouse Zoo blot probe recognizes a single human
RYK exon resistant to cleavage by HindIII within
the limits of homology to the probe. Thus it is likely that the signals
at 3.2 and 4.3 kb (Fig. 7a, human) reflect hybridization to
human RYK and its pseudogene. Hybridization to
RYK-related sequences in the human genome is also evident as
weaker signals (e.g. at ~1.8 kb; Fig. 7a).
Mapping of exon-intron boundaries of the zebrafish ryk gene
predicts the mouse Zoo blot probe to detect only one fragment, such
that the two strong zebrafish hybridization signals (Fig.
7a) can be confidently assigned to different loci.
Hybridization of the kinase domain probe to distinct genomic DNA
fragments was observed in all species tested, with the exception of
Escherichia coli (negative control). After prolonged
exposure, three autoradiographic signals were visible from
Drosophila genomic DNA (not shown). Ryk gene
orthologues and paralogues, both characterized and uncharacterized,
therefore seem to be widely conserved over the phylogenetic scale, from
mammals (human, mouse, horse, dog, cow, sheep, and rabbit), birds
(chicken), through lower vertebrates (Xenopus, zebrafish,
and carp), to invertebrate metazoans (Drosophila and
C. elegans).
We have determined in detail the genomic organization of the mouse
member of the growing subfamily of growth factor receptors related to tyrosine kinases
(Ryk). Although all subfamily members are currently orphan
receptors, RYK molecules are expected to function in the transduction
of growth-regulatory information across the plasma membrane by virtue
of their prototypical RTK topology, as has been demonstrated for all
other RTK subfamilies (5).
The mouse Ryk transcription unit resides on chromosome 9 (39, 50) and spans a minimum of 81 kb of genomic DNA. A large first
intron is a feature of the mouse Ryk gene which is shared with zebrafish ryk and Drosophila derailed (4). A
comparison of mouse Ryk with the structures of other
mammalian RTK genes shows that a large first intron is not uncommon
(e.g. human INSR, >25 kb, Ref. 34; human
IGF1R, >20 kb, Ref. 58; human FMS, 26 kb, and
human c-KIT, 37.4 kb, Ref. 59; human C-MET, 26 kb, Ref. 60). The GXGXXG motif vital for ATP
binding in PTKs is modified to XXGXXG in the RYK
subfamily. This motif is invariably encoded by a single exon; however,
in the mouse and C. elegans Ryk genes a type I splice donor
site interrupts the codon for the second consensus glycine residue, and
a type O splice site separates the fourth and fifth residues of motif I
in the zebrafish ryk gene (data not shown). Other mouse RYK
PTK subdomains, with the exception of IX, are encoded by single exons,
as is usually the case for other RTK genes. This supports the view that
the RYK kinase-like domains from phylogenetically diverse species are
evolutionarily related.
We speculate that the significance of exon splitting of RYK subdomains
I and IX, together with unusual kinase subdomain sequences, may be that
the ancestral Ryk gene arose very early in metozoan evolution and has since been subjected to selective pressure to maintain a modified PTK activity that relies heavily on atypical residues at normally conserved positions. Molecular modeling of the
mouse RYK PTK domain indicates that the nucleotide-binding cleft is
particularly large and may therefore indicate a preference for a
phosphodonor substrate other than
ATP.5 This
prediction is currently being tested.
Primer extension analysis performed by Yee et al. (40)
allowed us to identify the transcription start site at nucleotide 1627, within the CpG island (Fig. 2b). This result is consistent with the finding that almost every widely expressed gene transcribed by
RNA polymerase II that has been examined, such as mouse Ryk, has a CpG island at the 5' end that includes the transcription start
site (Refs. 31 and 44; CpG island data base
4.0).6 Assignment of the
transcription start site leads us to predict the synthesis of two mouse
Ryk mRNA species of 2.3 and 2.6 kb. Two polyadenylation
sites in exon 15, which appear to be utilized at equal frequencies in
most tissues (2, 37-41), define the predicted alternative 3'-mRNA
ends. By using RT-PCR, we can find no evidence for alternative splicing
of the Ryk primary transcript in 3T3 L1
cells.7 Wide variation in the
reported lengths of mouse Ryk transcripts could be due to
the inconsistent use of RNA size standards (i.e. RNA ladders
versus 28 S and 18 S rRNA species) in different
laboratories. The most accurate sizing of Ryk transcripts
seems to have been reported by Maminta et al. (61), where an
RNA ladder has been used to estimate mRNA lengths of 2.1 and 2.6 kb. These correspond well with Ryk mRNA sizes predicted
here by identification of the transcription start site and alternative
polyadenylation sites. However, the possibility that extensive and
stable secondary structure in the G + C-rich 5' end of the
Ryk mRNA, which survives denaturing conditions to
variable extents according to the particular methods employed by
different laboratories, remains an alternative explanation for the
apparent distortion of transcript lengths relative to RNA standards.
The G + C-rich nature of the 5' end of the mouse Ryk
mRNA is a common characteristic of transcripts encoding
growth-regulatory proteins. The likely mRNA secondary structure has
been proposed to function in the attenuation of translation as an extra
level of gene regulation (62). This region of the Ryk gene
is also highly enriched for the CpG dinucleotide relative to the bulk mammalian genome, where CpG depletion to 20% of the frequency expected
from base composition has resulted from methylation of CpG and the high
mutability of 5MeCpG to TpG or CpA (CpG suppression; Refs.
48 and 49). CpG dinucleotides in the Ryk promoter are
protected from genomic DNA methylation and are present at the frequency
predicted by base composition alone (i.e. no CpG
suppression). These properties identify the sequence spanning exon 1 of
the mouse Ryk gene as a CpG island 859 bp in size (Fig.
3).
A transcriptionally active CpG island represents a domain of "open"
chromatin structure characterized by core histone underacetylation, histone H1 depletion, and a nucleosome-free region (63). Constitutive binding of transcription factors is essential for the maintenance of a
methylation-free CpG island (46, 64). Virtually all sequenced genes
with widespread expression patterns are associated with a CpG island
(Ref. 44; CpG island data base
4.0),8 and mouse
Ryk can now be added to this class of genes. The human RYK gene is likely to be marked by a CpG island given that
approximately 80% of islands are common to mouse and man (65) and
human RYK also shows widespread expression of an mRNA
with a G + C- and CpG-rich 5' domain (3, 57). Other RTK genes with
known CpG islands include mouse and human FGFR3 (66), human
EGFR (67), human NTRK1/TRKA (Ref. 68; 423 bp CpG
island in exon 1 detected with CpGplot; data not shown), human
FLT1/VEGFR1 and FLT3 (69), human and mouse
KIT (59), and the human PDGF Our immunohistochemical analysis of mouse RYK further defines the
expression pattern of this unusual receptor. Whereas Northern blot and
RNase protection assays of Ryk mRNA indicate near
ubiquitous expression (2, 38-41, 55, 56), Wang et al. (57)
have reported localization of RYK mRNA to the epithelial
and stromal compartments of human tissues such as ovary, brain, lung,
colon, kidney, and breast by in situ hybridization. The
immunohistochemical staining results presented here confirm
localization of the functional Ryk gene product to the
tubular epithelium in kidney and to the stromal compartment of the
large intestine and indicate that mouse RYK is localized to the stroma
and epithelium of villi in the small intestine. However, organs
including embryonic day 14 heart and adult liver show strong and
homogenous expression of RYK throughout. Furthermore, the spatially
restricted localization of mouse RYK to distinct parenchymal
compartments of organs such as the adrenal gland and spleen suggests
that specific differentiated cell types not related in embryonic origin
may require the signal transduced by RYK.
We have demonstrated the existence of multiple Ryk subfamily
members in the mouse genome by Southern blotting: the canonical Ryk (2), a likely partial retroprocessed pseudogene derived from Ryk kinase domain exons, and a Ryk-related
gene detectable at quantitatively reduced stringency using
Ryk cDNA-derived probes to the extracellular and PTK
domains. The human genome appears to share with the mouse the feature
of a Ryk pseudogene, as well as the Ryk-related
gene. We have detected Ryk subfamily-like sequences in the
genomes of mammals, chicken, fish, and Drosophila by reduced stringency Southern blotting and in a nematode worm by cDNA
amplification and sequencing. Although the stringency conditions used
for Southern blot hybridization were relaxed, more stringent and
extensive washing was performed, suggesting that the level of
cross-species nucleotide sequence conservation in the kinase-like
domain is high.
Southern blot analysis of the Danio rerio (zebrafish) genome
showed hybridization signals representing the ryk locus plus a related sequence of unknown identity. From the simple nematode worm
C. elegans, we have sequenced a Ryk cDNA
orthologue, Ceryk, which predicts a transmembrane protein
demonstrating structural conservation of RYK subfamily-specific
features. These include a compact extracellular domain containing a
putative basic protease cleavage site flanked by universally conserved
cysteine residues. Post-translational processing of the human RYK
exodomain into The RYK proteins are structurally unique in at least two significant
features. Few recognizable protein motifs are present in an unusually
short extracellular domain, and phylogenetically conserved
intracellular substitutions in at least two highly conserved PTK
sequence elements responsible for cooperatively binding the Mg2+-ATP phosphate donor complex at the active site may
abrogate, or more likely modify, catalytic activity. These changes
perhaps reflect involvement of RYK in a unique cell-surface signal
transduction complex, where it may interact with a novel family of
extracellular ligands and/or be recruited into a heterodimeric
PTK-competent receptor complex. Alternatively, RYK could conceivably
function to attenuate signaling from such a complex by failing to
transactivate the protein tyrosine kinase activity of the partner
receptor in competition with homodimeric receptor formation. Third,
atypical substrate contacts mediated by the nucleotide binding cleft of RYK subfamily proteins, comprising variant Hanks' motifs I and VII,
may indicate an altered specificity for the phosphate, sugar, and/or
divalent cation moieties of the phosphodonor complex, as proposed by
Kelman et al. (39). We are further investigating the
function of RYK in embryonic and early postnatal development, and its
potential role in cancer, through the generation and analysis of
Ryk-deficient mice and a screen for ligands of the RYK receptor.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]dATP (Bresatec Ltd., Adelaide,
Australia; 10 µCi/µl, specific activity 3000 Ci/mmol) using a
Megaprime kit (Amersham, Buckinghamshire, UK) and purified. Denatured
probe was added to the buffer at 0.1-5 ng/ml and hybridization left to
proceed overnight. Oligonucleotide probes were end-labeled in 20-µl
reactions containing 100 ng of oligonucleotide, 1× polynucleotide
kinase (PNK) buffer, 5 mM dithiothreitol, 5 µl of
[
-32P]ATP (Bresatec Ltd., 10 µCi/µl, specific
activity 4000 Ci/mmol), and 5-10 units of PNK (MBI Fermentas, Vilnius,
Lithuania) at 37 °C for >30 min and purified. High stringency
washing involved three 5-min room temperature washes with 2× SSC,
0.1% SDS followed by two 20-30-min washes in 0.1× SSC, 0.1% SDS at
65 °C (genomic DNA Southern blots), or one 3-15-min wash in 6×
SSC, 0.1% SDS at 40-50 °C (oligonucleotide-probed Southern blots).
Blots were subsequently exposed to x-ray film (XAR-5 or BioMax MR;
Eastman Kodak Co.) with intensifying screens at
70 °C.
Ts =
21 °C) and a final washing stringency
of 0.6× SSC, 0.1% SDS at 50 °C for 30 min (twice).
RESULTS
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Fig. 1.
Organization of the mouse Ryk
gene (drawn to scale). a, exon-intron
distribution. Translated sequences are shown as black boxes
and untranslated sequences as open boxes. b,
partial restriction map of the Ryk locus. c,
overlapping recombinant BAC and lambda genomic DNA clones.
E, EcoRI; H, HindIII;
X, XbaI.
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Fig. 2.
Structure and sequence of the 5' region of
the Ryk gene. a, restriction map of
the 4-kb XbaI fragment subcloned from BAC.Ryk.2.
Sites for rare-cutting enzymes recognizing CpG-containing
hexanucleotide sequences are shown in boldface. The 0.7-kb
intron 1-derived probe used in Southern blotting is indicated. The
BssHII site in brackets is either incompletely
methylated or polymorphic in genomic DNA from W9.5 ES cells (see text
and Fig. 3b). b, DNA sequence of the region
surrounding exon 1. The boundaries of a CpG island are indicated by
curved arrows and a TATA box plus other putative
transcription factor binding motifs, with core sequences
underlined, are boxed. Only the single best
potential binding motif for each of the transcription factors shown is
boxed (core similarity >0.70, matrix similarity >0.8; see
"Experimental Procedures"). The transcription start site is marked
by a square arrow, and the antisense 35-mer used for primer
extension by Yee et al. (40) is represented by a
half-arrow, and alternative translation initiation
methionines (M1 and M2) are circled. The exon 1, 3' splice
donor site (type I) is boldfaced. SRF,
SRE-binding factor; NRF-2, nuclear respiratory factor 2;
ISRE, interferon-stimulated response element;
Tal-1, T-cell acute lymphocytic leukemia protein 1;
ER, estrogen receptor; STAT, signal transducer
and activator of transcription; AML-1, acute myeloid
leukemia protein 1; T3R, thyroid hormone receptor;
HNF-3 , hepatocyte nuclear factor 3
; IRF-1,
interferon regulatory factor 1; EGR, early growth response
protein; AhR/ARNT, aryl hydrocarbon receptor/aryl
hydrocarbon receptor nuclear translocator; RREB-1,
Ras-responsive element binding protein 1; NGFI-C, nerve
growth factor-induced protein C; Gfi-1, growth factor
independence protein 1.
Sequences around splice sites of the mouse Ryk gene
+c+p, Jackson Laboratory stock number
JR0090; Ref. 45), which express Ryk mRNA, was subjected
to Southern blot analysis with methylation-sensitive restriction
enzymes. Digestion with XbaI, or double-digestion with
XbaI and either BssHII or EagI (both
of which are blocked by methylation of either of the two CpG
dinucleotides in each of their recognition sequences (43)), was used to
assess the methylation status of four CpG dinucleotides within the
putative island. When XbaI-digested DNA was hybridized with
the 0.7-kb EcoNI-XbaI probe (see Fig.
2a), the expected 4-kb XbaI fragment was detected
(Fig. 3b). However, this fragment was not present in
doubly-digested DNA, demonstrating the presence of internal, unmethylated sites cleavable by BssHII and EagI.
Although the 0.7-kb probe hybridized with the expected 2.2-kb
BssHII-XbaI fragment, an additional 1.4-kb
BssHII-XbaI fragment was detected. This smaller fragment is generated by cleavage at a BssHII site 1.4 kb
upstream of the most 3' XbaI site (Fig. 2a) and
reflects either clonal variation in methylation of CpGs in the sequence
5'-GCGCGC-3', as previously reported for CpGs in DNA flanking the mouse
liver aprt CpG island (46), or an allelic polymorphism.
Allelic variation in simple sequence length polymorphisms has been
reported in the 129 mouse substrains and ES cell lines derived from
them (47), demonstrating that alleles are still segregating in these
supposedly highly inbred mice. As judged by the status of four CpG
dinucleotides within the Ryk CpG island, this region of DNA
is unmethylated and is expected to constitutively maintain this
property in expressing and nonexpressing cell types at all stages of
development (48, 49). The unmethylated or variably methylated status of
the BssHII site suggests that CpG dinucleotides immediately
surrounding the island may also be protected from methylation.
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Fig. 3.
Characterization of a CpG island at the 5'
end of Ryk. a, plot of the
observed/expected (O/E) ratio of CpG dinucleotide frequency
and G + C content versus nucleotide position as defined in
Fig. 2b. The sequence limits of the island and the relative
position of exon 1 are indicated. b, analysis of DNA
methylation at the Ryk CpG island by Southern blotting of
genomic DNA isolated from W9.5 ES cells. The origin of the 0.7-kb probe
used is shown in Fig. 2b. X, XbaI;
B, BssHII; E, EagI.
,
GATA-1, Nkx-2.5, MyoD) signals. This is consistent with the likely
function of RYK as a cell-surface growth factor receptor or
co-receptor, the expression of which is expected to be under stringent
spatial and temporal control (see "Immunohistochemical Analysis of
RYK Expression in the Mouse," below).
Ryk). At reduced
stringency, hybridization signals not present at high stringency are
visible using both extracellular and kinase domain probes from the
Ryk cDNA (Fig. 4, b and d,
arrowheads). Using a v-erbB PTK domain probe,
this level of hybridization stringency has proven to be suitable for
discriminating between RTKs highly related to the probe sequence, such
as subfamily members, while more distantly related members of the PTK
superfamily are not detected (25, 53). The detection of DNA fragments
at reduced stringency with probes to the extracellular and kinase
domains of mouse Ryk further suggests that the hybridization
signals represent a genuine subfamily member. We predict that this
second class of fragments is derived from a novel mouse member of the
Ryk RTK subfamily. Screening of genomic DNA libraries
enriched for
Ryk, which we have found to be
under-represented in supposedly complete genomic DNA libraries, and
Ryk-related fragments is underway.
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Fig. 4.
Southern blotting of mouse strain 129/Sv
genomic DNA detects multiple Ryk-related
sequences. Duplicate Southern blots were hybridized with an
extracellular domain probe at high stringency (a) and
reduced stringency (b), or with a kinase domain probe at
high stringency (c) and reduced stringency
(d). Hybridization signals in addition to those
expected from mapping of the mouse Ryk locus are indicated
by arrows (at high stringency) or arrowheads (at
reduced stringency). The expected 8.5-kb XbaI signal in
a is only faintly visible at high stringency due to the
small size of exon 5.
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Fig. 5.
Specificity of the RYK-1 monoclonal antibody
and immunohistochemical analysis of RYK expression in the mouse.
a, Western blot of RYK-EX-FLAG probed with the RYK-1
antibody. A negative control lane containing RYK-EX-FLAG was probed
with supernatant from a hybridoma which scored negative in the enzyme
immunoassay screen. b, Western blot of alkaline
phosphatase-tagged human RYK and VEGFR2 extracellular domains probed
with the RYK-1 antibody. c, Western blot analysis of MCF-7
human breast carcinoma total cell lysate (105 cells) with
the RYK-1 antibody. The signal at 85,000-90,000 kDa
(arrowhead) represents the RYK receptor. Kidney
(d and e), liver (f and g),
adrenal gland (h and i), embryonic day 14 heart
(j and k), spleen (l and
m), small intestine (n and o), and
large intestine (p and q) were stained with RYK-1
(left column) or an isotype-matched (IgM) negative control
antibody, respectively. Scale bars, 100 µm.
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Fig. 6.
Deduced amino acid sequence of C. elegans RYK and alignment with other members of the RYK
subfamily of proteins. Alternative ATG translation start codons
are marked in the mouse sequence (*). N-terminal signal sequences and
their predicted sites of cleavage are shown ( ). The transmembrane
domains (TM), mammalian leucine-rich motifs
(LRMs), putative tetrabasic cleavage sites (TBC),
conserved cysteine residues (
), Hanks kinase motifs
(I-XI), potential autoregulatory tyrosine residue
homologous to that in pp60src (crossed
circle), boundaries of the protein tyrosine kinase domain (bent arrow), and the C-terminal, PDZ domain
Y(V/I)-COOH ligand consensus sequence (PDZL) are indicated. Residues
that match the sequence of mouse RYK are boxed.
Mm, Mus musculus; Hs, Homo
sapiens; dnt, doughnut; drl,
derailed; Ce, C. elegans.
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Fig. 7.
Phylogenetic conservation of Ryk
subfamily molecules. a, zoo blot of genomic DNAs
digested with HindIII and hybridized with a 159-bp probe
encoding mouse Ryk exon 13. The expected 3.6-kb
hybridization signal is present in the lane containing mouse DNA, in
addition to a smaller fragment of ~700 bp, probably derived from a
pseudogene. All species gave rise to observable hybridization signals
(lane containing Drosophila DNA required longer exposure,
not shown) with the exception of E. coli (negative control).
The two strong signals in the zebrafish lane represent distinct genes
(see text). b, PCR analysis of human RYK exons
encoding sequences homologous to the mouse exon 13 probe. PCR primers
flanking the human cDNA sequence homologous to the mouse Zoo blot
probe amplify an intronless 159-bp product from human genomic DNA, not
cleavable by HindIII, which hybridizes to an internal
oligonucleotide (lower panel, 10-min exposure).
M, 100-bp ladder; G, human genomic DNA template;
, negative control (no template).
DISCUSSION
R (CpG island data base 4.0).8
disulfide-linked subunits analogous to the
c-MET/HGF receptor (70), mouse STK/RON (71), the insulin receptor (72),
and the insulin-like growth factor 1 receptor (73) is supported by our
Western blotting and immunoprecipitation data (Fig. 5c and
Ref. 3), although we have found human RYK to be inconsistently processed in this manner. In the predicted CeRYK protein, a protein tyrosine kinase-like domain with strongest sequence homology to other
RYKs, distinctive RYK-specific amino acid substitutions in PTK
subdomains I and VII (11), and a C- terminus conforming to the
consensus -Y(V/I)-COOH, representing a possible PDZ domain ligand (74),
are identifiable in the intracellular domain (Fig. 6). No likely
candidate C. elegans mutant phenotypes mapping to this
CELC16B8 chromosomal locus are known.
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ACKNOWLEDGEMENTS |
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We thank Dr. Guo-Fen Tu for DNA sequencing, Dr. Andrew Runting for assistance with computing, Cuong Do for access to unpublished data, and Profs. Ashley Dunn and Antony Burgess for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Cooperative Research Center for Cellular Growth Factors.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) AF080624 for the putative mouse Ryk promoter.
Recipient of an Australian Postgraduate Research Award.
§ Recipient of an Anti-Cancer Council of Victoria Postgraduate Research Award. Current address: Dept. of Molecular Biology, Princeton University, Washington Rd., Princeton, NJ 08544.
¶ Supported by a Senior Research Fellowship from the Australian Research Council.
Current address: Mutation Research Centre, Daly Wing, St
Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
** To whom correspondence should be addressed. Tel.: 613-9341-3130; Fax: 613-9341-3107; E-mail: Steven.Stacker{at}ludwig.edu.au.
2 Oates, A. C., Bonkovsky, J. L., Irvine, D. V., Thomas, J. B., and Wilks, A. F. (1998) Mech. Dev. 78, 165-169.
3 C. P. Do, A. C. Oates, and A. F. Wilks, manuscript in preparation.
4 J. L. Bonkovsky, personal communication.
5 H. Treutlein, M. M. Halford, and S. A. Stacker, unpublished data.
6 Available on-line at the following address: ftp://ftp.no.embnet.org/cpgisle/relnotes4.0.doc.
7 M. M. Halford and S. A. Stacker, unpublished data.
8 Available on-line at the following address: ftp://ftp.ebi.ac.uk/pub/data bases/cpgisle/.
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ABBREVIATIONS |
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The abbreviations used are: RTK, receptor-type tyrosine kinase; Ryk, receptor related to tyrosine kinases; RYK-EX-FLAG, FLAG-tagged human RYK extracellular domain; PTK protein tyrosine kinase, EGFR, epidermal growth factor receptor; VEGFR, vascular endothelial growth factor receptor; FGFR, fibroblast growth factor receptor; INSR, insulin receptor; LRM, leucine-rich motif; BAC, bacterial artificial chromosome; RT-PCR, reverse transcriptase-polymerase chain reaction; nt, nucleotide; kb, kilobase pair(s); bp, base pair(s).
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REFERENCES |
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