From the New England Baptist Bone and Joint
Institute, Beth Israel Deaconess Medical Center, Boston, Massachusetts
02115, § Harvard School of Dental Medicine, Boston,
Massachusetts 02115, and
Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, August 6, 2000, and in revised form, March 12, 2001
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ABSTRACT |
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The gene encoding the murine
calcitonin receptor (mCTR) was isolated, and the exon/intron structure
was determined. Analysis of transcripts revealed novel cDNA
sequences, new alternative exon splicing in the 5'-untranslated
region, and three putative promoters (P1, P2, and P3). The
longest transcription unit is greater than 67 kilobase pairs, and the
location of introns within the coding region of the mCTR gene (exons
E3-E14) are identical to those of the porcine and human CTR genes. We
have identified novel cDNA sequences that form three new exons as
well as others that add 512 base pairs to the 5' side of the
previously published cDNA, thereby extending exon E1 to 682 base
pairs. Two of these novel exons are upstream of exon E2 and form a
tripartite exon E2 (E2a, E2b, and E2c) in which E2a is utilized by
promoter P2 with variable splicing of E2b. The third new exon (E3b')
lies between E3a and E3b and is utilized by promoter P3. Analysis of mCTR mRNAs has revealed that the three alternative promoters give rise to at least seven mCTR isoforms in the 5' region of the gene and
generate 5'-untranslated regions of very different lengths. Analysis by
reverse transcription-polymerase chain reaction shows that promoters P1
and P2 are utilized in osteoclasts, brain, and kidney, whereas promoter
P3 appears to be osteoclast-specific. Using transiently transfected
reporter constructs, promoter P2 has activity in both a murine kidney
cell line (MDCT209) and a chicken osteoclast-like cell line (HD-11EM),
whereas promoter P3 is active only in the osteoclast-like cell line.
These transfection data confirm the osteoclast specificity of promoter
P3 and provide the first evidence that the CTR gene is regulated in a
tissue-specific manner by alternative promoter utilization.
The calcitonin receptor
(CTR),1 which contains seven
transmembrane domains, is a member of the class II G protein-coupled
receptor family (1, 2). The class II family, while structurally
related, has little similarity at the amino acid level to the class I
family (e.g. rhodopsin and The CTR gene has a complex structural organization with several CTR
protein isoforms derived from alternative splicing of transcripts from
a single gene (15). These isoforms, which are functionally distinct in
terms of ligand binding specificity and/or signal transduction pathway
utilization, are distributed both in a tissue-specific and
species-specific pattern (16-27). Furthermore, spliced mRNA
products have been identified in hCTR that generate translation
terminations shortly after transmembrane domain 1 (28, 29),
resulting in the expression of truncated CTR proteins.
Although some of the human (23) and porcine CTR (pCTR) (22) genomic
sequences have been cloned, little is known about the mechanism of
transcriptional regulation for the CTR gene in osteoclasts or in other
tissues in which it is expressed. A 657-bp fragment of the pCTR
promoter was demonstrated to drive expression of a luciferase reporter
gene when transfected into the CTR-expressing porcine kidney epithelial
cell line LLC-PK1 (22). Recently, the function of a 2.1-kb
fragment of the pCTR promoter has been assessed in a transgenic mouse
by Jagger et al. (30). They found that although this region
directed expression of the lacZ reporter in several
embryonic and fetal tissues that express endogenous mCTR, it was not
sufficient to direct transcription in the adult kidney or bone of the
transgenic mice.
In this report, the gene encoding the murine CTR (mCTR) was isolated,
and the exon/intron structure was determined. We have identified novel
cDNA sequences that extend the beginning of the previously
published mCTR cDNA (21) by 512 bp, thereby enlarging exon E1 to
682 bp. Further analysis of mCTR cDNAs has also revealed three new
exons within the 5'-UTR, new alternative exon splicing in the 5'-UTR,
and the presence of three putative promoters (P1, P2, and P3). Two of
these novel exons are upstream of the original cDNA exon E2 and
form a tripartite exon E2 (E2a, E2b, and E2c) in which E2a is utilized
by promoter P2 with variable splicing of E2b. The third new exon (E3b')
lies between E3a and E3b and is utilized by promoter P3. Analysis of
mCTR mRNAs reveals that the transcripts from the three promoters
are spliced to yield seven different 5'-UTR structures. Analysis by
both RT-PCR and transient transfection of promoter-luciferase reporter
constructs shows that the P1 promoter (located upstream of an expanded
exon E1) and the P2 promoter (located upstream of exon E2a) are
utilized in osteoclasts, brain, and kidney, whereas the P3 promoter
(located upstream of the novel exon E3b') appears to be exclusively
utilized in osteoclasts. The P2 promoter of mCTR is highly homologous
to the promoter region previously defined for pCTR in kidney cells. These studies provide the first evidence that CTR is regulated in a
tissue-specific manner by alternative promoter utilization and that
there is a unique promoter (P3) that regulates CTR expression only in osteoclasts.
Isolation and Characterization of the Mouse CTR Gene--
A
murine genomic library made from the 129 mouse strain in Lambda FIX II
(provided by Dr. Chuxia Deng, Bethesda, MD) was screened using probes
from the murine CTR cDNA clone isolated by Yamin et al.
(21). The probes were labeled with 32P by random priming.
The 13 positive phage clones were plaque-purified, and DNA was prepared
utilizing standard procedures. NotI fragments containing the
mCTR genomic DNA from these phage were subcloned into pBluescript KS
(Stratagene) and analyzed by restriction enzyme site mapping and
hybridization with region-specific CTR probes. In some cases, PCR
between exons was used to determine the intron size. Note that the
originally published cDNA (21) included 14 bp of adapter and the
mCTR sequence actually starts at position 15 (GenBankTM
accession number U18542).2
Genomic regions of interest, such as exon/intron junctions, were sequenced using the dideoxy chain termination method (31) with Sequenase polymerase version 2 (U.S. Biochemical Corp.) and primers based on previously known or novel mCTR sequence as necessary. The
exon/intron junctions were established by comparison of genomic sequence with the cDNA sequence. Comparison with other CTR genomic sequences was done using both BLAST (32) and the GCG sequence analysis package (33).
RNA Preparation--
Total RNA was isolated from either mouse
organs (brain, liver, kidney), osteoclast cocultures, or cell lines by
using Trizol Reagent (Life Technologies, Inc.) according to the
manufacturer's instructions. Osteoclasts were generated by coculture
of hematopoietic progenitors with the stromal cell line ST2 essentially
as previously described (34). Bone marrow cells were collected by
flushing the femurs and tibiae of 5-week-old male C57/Bl6 mice with
Characterization of the 5'-End of Exon E1 by Primer Extension and
Ribonuclease Protection Assay (RPA)--
RNA samples (5-20 µg) from
various tissues or cell lines were reverse transcribed to generate
primer extension products using the Ready-To-Go You-Prime First-Strand
Beads kit (Amersham Pharmacia Biotech) and an mCTR-specific antisense
primer located in exon E1 end-labeled with [
To determine the 5'-end of exon E1, an RPA was performed. A
532-bp mCTR genomic DNA fragment spanning from a BsaBI site
to a BamHI site encompassing the 5'-end region and part of
exon E1 was blunted with Klenow and subcloned into the SmaI
site of pBKS vector, linearized with BamHI, and used as a
template for synthesizing a 635-bp riboprobe with
[ 5'-Rapid Amplification of cDNA Ends (5'-RACE)--
To
identify and verify the sequence of the 5'-ends of mCTR transcripts,
mouse brain, kidney, and osteoclast RNA samples were used to generate
adapter-ligated double-stranded cDNA using the Marathon cDNA
amplification kit (CLONTECH). For each PCR
reaction, 2 µl of the double-stranded cDNA adapter ligation
solution diluted to 1:10 was added into a total volume of 50 µl of
PCR buffer solution containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 1 µM each of the 5' and 3' primers, 2 mM dNTPs,
and 2.5 units of Taq DNA polymerase (Fisher). Several
5'-RACE experiments were performed using different primer sets with two
rounds of PCR. The first round PCR was performed using the sense
adapter primer AP1 and two different mCTR antisense primers, either
mPE1 or E6-R (see Table I). The AP1 + mPE1 PCR products were
used for a second nested round of PCR using the sense adapter primer
AP2 and the mCTR primer mPE3. The AP1 + E6-R PCR products were used for
several nested PCRs using sense primer AP2 and either mCTR antisense
primer E4-R, E5-R, or E6-R. The PCR conditions for both PCR rounds were 94 °C for 15 s, 60 °C for 15 s, and 68 °C for
15 s for 30 cycles using the Gene Amp PCR system 9600 (PerkinElmer
Life Sciences). Southern blot analysis was performed using
[ Tissue Distribution of mCTR Isoforms Derived from Three Promoters
Using RT-PCR--
For reverse transcription reactions, 2 µg of total
RNA samples from mouse liver, brain, kidney, and osteoclasts derived
from bone marrow cocultures were used to generate cDNAs with 0.5 µg of oligo(dT) using the reverse transcription Ready-To-Go You-Prime First-Strand Beads kit (Amersham Pharmacia Biotech). Different sets of
mCTR-specific primer pairs were used to analyze the variable structure
of the mCTR mRNAs, and a pair of primers specific for GAPDH was
used to assess the quality of the RNA samples. Additionally, a pair of
primers (mTM5-F and mTM7-R) from a mCTR region without variable
splicing was used to establish the presence or absence of mCTR mRNA
in each sample. For all PCRs except GAPDH and plasmid controls, equal
amounts of each cDNA (2 µl of a 30-µl RT reaction) were added
to a total volume of 50 µl of PCR solution similar to that previously
described in the 5'-RACE method. For the GAPDH PCR, only 1 µl of the
RT reaction was added to the PCR. PCR of relevant mCTR cDNA
plasmids (1 ng) generated by the 5'-RACE (P1.1, P2.1, P2.3, and P3.1)
were used to prepare control PCR products. Also, for all PCR reactions,
a negative control containing H2O instead of cDNA was
run. The PCR conditions for the 110-bp GAPDH PCR product were 95 °C
for 2 min and then 30 cycles of 95 °C for 30 s and 60 °C for
30 s, ending with 72 °C for 7 min. To analyze the mCTR mRNA
splice isoforms generated from the P1, P2, and P3 transcripts, either
sense primer E1-F, E2a-F, or E3b'-F was used, respectively, together
with antisense primer E4-R. PCR conditions were 94 °C for 15 s,
60 °C for 15 s, and 72 °C for 30 s for 35 cycles,
except for E2a-F2 + E4-R, for which 50 °C was used instead of
60 °C. Southern blot analysis was performed sequentially or on
parallel blots with [ Construction of the P2 and P3 mCTR-pGL3 Luciferase
Vectors--
To test the putative P2 and P3 mCTR promoter activities,
the blunt-ended mCTR DNA genomic fragments containing appropriate P2
and P3 regions were subcloned in both orientations into the SmaI site of the pGL3 basic luciferase vector (Promega),
which does not contain a promoter or an enhancer. The initial P2
genomic region used spanned from the BamHI site in E1
( Transient Transfections--
The promoter activity of the
various P2- and P3-pGL3basic constructs were tested in two different
cell lines, a murine distal convoluted tubule cell line (MDCT209) (36)
(a generous gift from Dr. P. Friedman, Dartmouth, Hanover, NH) and the
chicken osteoclast-like cell line HD-11EM (37, 38) (kindly provided by
Dr. P. Hauschka, Boston, MA). The cell lines were maintained in
Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 5% (MDCT209) or 10% (HD-11EM) fetal calf serum. Briefly, 2 × 105 cells/well (MDCT) or 1 × 106
cells/well (HD-11EM) in a six-well plate were incubated at 37 °C
overnight. The P2-pGL3 and P3-pGL3 plasmids (1.5-3 µg/well) were
transiently transfected (in triplicate) into each cell line in
serum-free medium using 5-12 µl LipofectAMINE (Life
Technologies, Inc.) and incubated 4-5 h before an appropriate amount
of serum was added, and the cells were further incubated at 37 °C
for 24-48 h. The transfected cells were harvested by washing twice
with phosphate-buffered saline (pH 7.4) followed by incubation with 300 µl of lysis buffer (25 mM glycyl glycine buffer (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1% Triton
X-100, and 1 mM dithiothreitol) for 30 min at 4 °C.
Luciferase activities were determined in luciferase buffer (25 mM glycyl glycine buffer (pH 7.8), 15 mM
KPO4 (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, and 1 mM
dithiothreitol) using an AutoLumat (EG Structure of the mCTR Gene--
A murine genomic library was
screened using probes derived from a previously isolated mCTR cDNA
clone (21). We obtained 13 positive phage clones (Fig.
1A) encompassing most of the
transcription unit (>67 kb) with the exception of part of one very
long intron (IVS3b') that is greater than 13.9 kb. In addition, the
genomic DNA corresponding to a gap between the sequences contained in phages 3 and 15 was isolated by using PCR between exons E6 and E7
employing mouse strain 129 genomic DNA as template (CD-PCR in Fig. 1A). The identified phage clones include 15 kb of
the putative 5'-flank and 8 kb of the 3'-flanking regions.
Characterization of all the exon/intron junctions, determination of
intron sizes, and mapping of sites for multiple restriction enzymes is
shown in Fig. 1A. Sequence analysis confirms the presence of
appropriate donor-acceptor consensus sequences (GT-AG) (39, 40)
at the ends of each intron (Table II). A schematic of the relationship between the exon borders and the protein regions is depicted in Fig.
1B. Of interest, the location of the exon/intron junctions of the coding exons in the mCTR gene are exactly the same as in the
porcine and human CTR genes (22, 23). This includes the two putative
translation start sites (Fig. 1A, labeled aa)
that are split between two exons in the murine (E3a and E3b) and human (71-bp insert and E3) CTR genes. Use of the upstream translational start would add 17 amino acids to the mCTR protein (Fig. 1B,
shaded box at the N terminus of the protein).
Exon E8b is a rodent-specific 111-bp coding exon that adds 37 amino
acids to the extracellular domain 2 of mCTR and is alternatively
spliced in mCTR mRNAs (18, 21, 41, 42). A number of sequence
conflicts were found between our genomic sequence and our originally
published mCTR cDNA (21) in exons E1, E2c, and E8b. The newly
isolated cDNAs described below contain the same E1, E2c, and E8b
sequences as the genomic sequence.
Identification of the 5'-End of Exon E1--
In order to determine
the transcription start site of the mCTR gene, primer extension and
5'-RACE analyses were performed using RNA from mouse tissues known to
express CTR and in which CT exhibits biological activities (murine
brain, kidney, and osteoclasts). The primer extension analysis was
first carried out with a 32P-end-labeled mCTR-specific
antisense primer (mPE1; see Fig.
2A) with its 3'-end located 75 nt downstream from the reported 5'-end of mCTR (21). The results showed
that for all the tissues there were several primer extension products
distributed more than 300 bp beyond the original 5'-end (data not
shown). Since the primer extension results indicated that the 5'-end of
the mCTR mRNA is located upstream of the 5'-end previously
reported, 5'-RACE was performed to isolate the predicted additional
cDNA sequences. The mCTR-specific primer used for the final PCR
step was mPE3 (see Table I and Fig.
2A). The subcloned products containing mCTR inserts were
identified by hybridization with 32P end-labeled mPE4
probe. Representative 5'-RACE clones with varying insert sizes from
each of the RNAs were sequenced and compared with mCTR genomic DNA as
shown in Fig. 2A. This reveals that all of the newly
identified 5'-cDNA sequences are contiguous with the previously
defined exon E1 in the mCTR genomic DNA, thereby only extending E1
without forming a new exon. The clones generated from mouse brain
RNA contained an additional 56, 66, 240, and 372 nt (Fig.
2A, *); those from kidney RNA contained an additional 10 nt
(Fig. 2A, +); and those from osteoclast RNA contained an additional 56, 66, and 84 nt (Fig. 2A,
To clarify whether any of these new cDNA sequences include the true
5'-end of mCTR mRNA, another primer extension analysis was
performed using antisense primer mPE6 (see Fig. 2A) with its 3'-end located 156 nt downstream of the new putative mCTR 5'-end. The
results from this primer extension analysis of RNAs from murine brain,
kidney, and osteoclasts revealed several products longer than the new
putative 5'-end identified above by ~150, 170, 300, and >300 nt
(data not shown). Even with repeated 5'-RACE, we have been unable to
recover the additional 5'-cDNA sequences revealed by mPE6 primer
extension. Therefore, to determine the 5'-end of exon E1, an RPA was
performed. A subcloned 532-bp mCTR genomic DNA fragment containing 430 bp of putative 5'-genomic sequence and 102 bp of exon E1 (as
defined by 5'-RACE) DNA was used as a template for synthesizing a
635-bp [ Isolation of Novel mCTR cDNAs Indicating the Presence of Two
Novel Primary Transcripts--
Analyses of the paralogous genes coding
for mouse and rat PTH/PTHrP-R have revealed that there are
multiple transcription initiation sites regulated by different
tissue-specific promoters (43-46). To search for the existence of
splicing isoforms of the 5'-end of the mCTR gene, additional 5'-RACE
was performed in which the first PCR step employed primers AP1 and E6-R
(an antisense primer located in exon E6) (see Table I). The product of
this reaction was analyzed by Southern hybridization, cloning, and sequencing. It was also used in a second round of PCRs with three different primer pairs: AP2 + E4-R, AP2 + E5-R, and E1-F + E5-R (see
Table I). Sequence analysis of representative cDNA clones generated
from osteoclast RNA revealed four different mCTR cDNAs. These are
apparently derived from two primary transcripts that initiate from two
novel exons designated E2a and E3b' and do not include exon E1 (Fig.
3). The primary transcript initiating
from exon E2a is spliced to form mRNAs, with three different 5'-UTR structures designated as P2.1, P2.2, and P2.3 in Fig. 3A.
Exon E2a was observed in the 5'-RACE products to contain an additional 10-12 bp that are 5' to the exon E2 sequence (redesignated E2c) found
in the previously reported mCTR cDNA. The P2.1 and P2.2 mRNA
isoforms differ by the presence or absence of exon E3a, which contains
the upstream putative translation start. In the mCTR P2.3 mRNA
isoform, an additional 182-bp exon (designated E2b) was identified
between exons E2a and E2c. This isoform was found to contain the
variably spliced exon E3a. The newly identified exons E2a and E2b do
not contain an ATG translational start codon and are probably 5'-UTR
sequences. Therefore, their inclusion in a mRNA does not change the
mCTR protein structure.
The primary transcript initiating from the novel exon E3b' is spliced
to form a mRNA, with a single 5'-UTR structure designated as P3.1
in Fig. 3B. Exon E3b' was found by 5'-RACE to contain 65 bp
that are located upstream of exon E3b sequences (Fig. 3B). Exon E3a, containing the upstream translation initiation codon, is not
present in the P3.1 type mRNA. Also, exon E3b' does not contain an
ATG codon. Therefore, translation from this mRNA probably starts in
exon E3b.
Analysis of genomic DNA by PCR between exons along with Southern
blotting with region-specific oligonucleotide probes revealed that the
novel exons are located within the mCTR genomic region defined with the
earlier cDNAs (see Fig. 1). Genomic regions around the new exons
were sequenced, including the putative promoter regions P2 (Fig.
4) and P3 (Fig.
5) upstream of exons E2a and E3b',
respectively (also see Table II). Exons
E2a and E2b were found to be contiguous in the genomic DNA with exon
E2c (Fig. 4). Therefore, exon E2b serves both as an exon in the P2.3
type mRNA and as an intron in the P2.1 and P2.2 type mRNAs. The
exon E2b sequence contains the GT-AG splice 5'-donor-3'-acceptor
consensus sequences required for it to be processed as an intron
between E2a and E2c, and it therefore may also be considered to be a
"retained intron." Intron retention is another form of alternative
splicing that has been observed to occur in a number of different genes such as Drosophila male-specific lethal 2 (47), bovine and
human growth hormone, murine vitamin D receptor, and rat renal outer medulla K+ channel (see Ref. 48 and references therein).
Exon E3b' is located 690 bp downstream of exon E3a and an unknown
distance (>12.9 kb) upstream of exon E3b (Figs. 1A and 5).
The sequences upstream of the putative 5'-ends of exons E2a and E3b' do
not contain a 3'-splice acceptor consensus sequence, suggesting
that these exons are probably used to initiate mCTR transcripts. In contrast, the sequences downstream of exons E2a and E3b' contain 5'-splice donor consensus sequences (see Table II and Figs. 4 and 5).
Primer extension and RPA were attempted to further define the 5'-ends
of the P2 and P3 transcripts. However, due to the small sizes of exon
E2a and E3b', the usage of E2c and E3b in mRNAs initiated upstream
of E2a and E3b', and the very low abundance of mCTR RNA, we have not
been able to establish the definitive 5'-ends. However, in light
of the sequence homology within the region around E2a between mCTR,
pCTR, and hCTR (see Fig. 9) and for the sake of consistency across the
species, we have assigned the +1 of E2a to be 21 bp upstream of the E2a
start detected by 5'-RACE (Figs. 3 and 4).
mCTR mRNA Isoform Tissue Distribution--
To assess the
presence of P1, P2, and P3 mCTR transcripts in mouse osteoclasts,
kidney, liver, and brain, semiquantitative RT-PCR was performed using
primers E1-F, E2a-F, or E3b'-F plus E4-R to separately detect all of
the mRNA products derived from each of the primary transcripts
through exon E4 (Fig. 6). The RT-PCR
cDNA products were hybridized sequentially or in parallel with
32P-labeled oligonucleotides from exons E2a, E2b, E2c, E3a,
E3b', and E3b in order to detect and confirm all of the isoforms.
Additional PCRs with primers from a region of mCTR included in all
mRNAs (mTM5-F + mTM7-R; see Table I) and primers for GAPDH were
used, respectively, to assess the presence of mCTR mRNA and the
general quality of the mRNA preparations and RT reactions. The
results of these analyses are shown in Figs. 6 and
7. The data observed in Fig. 6,
A and B, demonstrate that liver does not express
CTR. As observed earlier with the P2 mRNAs, exon E3a is alternately spliced in P1 mRNAs (Figs. 6C and 7). In osteoclast,
brain, and kidney, the predominant P1 transcript splice isoform appears
to be the P1.1, which contains exon E3a (+E3a). Although the P1.2 ( Function of the Putative P2 and P3 mCTR Promoters--
In order to
establish that the putative mCTR P2 and P3 promoters are functional,
luciferase reporter constructs containing these regions were
transfected into both the mouse kidney cell line MDCT209 and the
chicken osteoclast-like cell line HD-11EM (Fig.
8A). The full-length mCTR P2
region containing 1253 bp of sequence 5' to exon E2a (including a
portion of exon E1) as well as exons E2a, E2b, and E2c (+398) was
cloned into the pGL3basic vector (which lacks both heterologous
enhancer and promoter) in both the forward (
We further investigated which regions of the P2 promoter are important
for activity by deleting both from the 5' and the 3' sides of the most
active P2 mCTR-pGL3basic construct ( In the present study, we cloned and characterized the mCTR gene.
It contains 19 exons distributed over a region >67 kb long. This
report establishes that the mCTR gene is transcribed from three
different promoters in a tissue-specific fashion and that these three
primary transcripts are spliced within the 5'-UTR to generate seven
mRNA isoforms (see Fig. 7). Of note, a RNA transcript of ~4.2 kb
was identified in RNA from mouse kidney and brain using Northern blot
analysis (21), and the original mCTR cDNA (which is a type
P1.1 5'-UTR and contains E8b) is only 3736 bp. With the
additional novel 512 bp of exon E1 added, this isoform of the mCTR
mRNA is then expected to be 4247 bp. However, primer extension
results indicate that this mRNA isoform is even longer and that
there is another exon upstream of exon E1. RT-PCR products synthesized
from exon E1 to downstream exons such as E2c, E3, E4, etc. have never
been found to include exon E2a, E2b, or E3b' (see Fig. 6), thereby
adding weight to the argument that exons E2a and E3b' are only used to
initiate mCTR transcripts P2 and P3, respectively, and are always
spliced out of primary transcripts that start upstream. Rodent CTRs
contain an additional variably spliced exon (111-bp E8b) within the
coding sequence whose presence adds 37 amino acids to the extracellular
domain 2 and alters the ligand specificity (18, 21, 41, 42). We have
not established the configuration of the seven 5'-UTR splice forms with
the presence or absence of exon E8b. However, exon E8b is primarily
retained in mCTR mRNA expressed in the brain. Therefore, mCTR
mRNAs with 5'-UTR types P1.1, P2.1, P2.3, and P2.4 are the only
isoforms likely to contain exon E8b.
mCTR Genomic Structure--
Both the P2 and the P3 promoters lack
many well known transcription initiation site consensus sequences.
These include a TATA box in the
There are a number of potential transcription factor binding sites that
can be identified within both promoters, including many possible sites
for C2-H2 zinc finger type transcription
factors in P2 (57, 58). Nishikawa et al. (59) recently
isolated an hCTR cDNA containing the equivalent of E2a-E2c by
5'-RACE using a human mammary tumor cell line, MCF-7. Comparison of the
5'-UTR Lengths and Translation--
The utilization of three
alternative promoters that give rise to at least seven mCTR isoforms in
the 5' region of the gene with 5'-UTRs of very different lengths
(see Fig. 7) raises questions concerning the functional significance of
these heterogeneous mCTR transcripts. Determination of the exon/intron
structure of the mCTR gene reveals that the two putative in-frame
translation start sites are localized to two separate exons (E3a and
E3b). Both of these exons were included in the originally published mCTR cDNA (21), but alternative splicing of exon E3a is evident from the data presented in this report. The mCTR mRNA types P1.1, P2.1, and P2.3 contain E3a, whereas it is spliced out of types P1.2,
P2.2, P2.4, and P3.1. The translation start site used when both exons
E3a and E3b are present has not been established. However, both agree
well with the Kozak consensus (RXXAUGR) (62) for translation starts, and therefore, it is reasonable to presume that
when exon E3a is present, its AUG is used to initiate translation (63).
The translation product initiated in E3a is 17 amino acids longer than
that initiated in E3b, and these 17 amino acids are quite hydrophilic.
However, the 17 amino acids encoded by exon E3b, when translation
starts at that AUG, are very hydrophobic and probably constitute a
"signal peptide." When translation initiates in E3a, it is possible
that the N terminus of mCTR is cytoplasmic, with the adjacent sequence
of hydrophobic residues forming an additional eighth transmembrane
domain. Like mCTR, the hCTR mRNA has also been reported to contain
two putative translation start sites (17), and the upstream start is
contained within a variably spliced 71-bp exon (24). The translation
product initiated from the hCTR upstream start site is 18 amino acids
longer than if initiated from the downstream start site. The hCTR 71-bp
insert is located 8896 bp upstream of hCTR exon E3 in BAC
GS1-117O10 (GenBankTM accession number AC003078) at
positions 104875-104804.
The P1 mRNAs have very long 5'-UTRs of >955 and >898 nt (see Fig.
7) that are slightly GC-rich (53% GC) and contain seven AUGs before
the AUG in E3a. Most have a pyrimidine at
Osteoclasts express all seven 5'-UTR mCTR isoforms. However, the
mRNA isoforms containing E3a are more abundant than their counterparts lacking E3a. It has not been possible to quantitate the
relative promoter usage. On the other hand, in kidney and brain,
promoter P3 is not utilized, and it appears that the kidney does not
use exon E2b. The presence of alternative promoter usage and splicing,
localized to the 5'-end of the mCTR gene, may thus provide a mechanism
for regulating the expression of this gene at both the translational
and transcriptional level. The existence of an mCTR promoter (P3) that
is osteoclast-specific is, perhaps, not surprising due to the fact that
CTR is expressed in a restricted spectrum of tissues with developmental
regulation. Upon proper stimulation, the osteoclast precursor, which is
of monocyte/macrophage lineage, undergoes a program of cellular
differentiation in which a distinct profile of genes are induced,
including, for example, cathepsin K,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptor). The CTR
is coupled to multiple signal transduction pathways. Binding of the
32-amino acid peptide hormone calcitonin can stimulate activation of
the following: the adenylate cyclase/cAMP/protein kinase A pathway (3);
the phosphoinositide-dependent phospholipase C pathway (which results in Ca2+ mobilization (4) and protein kinase
C activation (5)); and the phosphatidylcholine-dependent
phospholipase D pathway (which also results in protein kinase C
activation) (6). Calcitonin directly inhibits bone resorption by
osteoclasts and enhances renal calcium excretion (7-9). It also has
effects on the central nervous, cardiovascular, gastrointestinal, and
reproductive systems, and CTRs have been identified on osteoclasts,
certain kidney cells, some regions of the brain, testis, ovary, and
spermatoza (for a review, see Ref. 10). Recently, it has been
demonstrated that the human CTR (hCTR), when coexpressed with receptor
activity-modifying proteins, is also a receptor for the 37-amino acid
peptide hormone amylin (11-13). This peptide has effects on insulin
release, glucose uptake, and glycogen synthesis in skeletal musculature
(14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium. The cells were cultured for
24 h in
-minimal essential medium containing 10% fetal calf
serum, and then the nonadherent population was collected and
erythrocytes were removed using a density gradient centrifugation on
Ficoll-Hypaque (Sigma). The remaining hematopoietic progenitors were
cocultured with ST2 cells at a 1:10 ratio in medium containing
1,25-(OH)2D3 (10
8
M) and dexamethasone (10
7
M). After 9 days of coculture, the ST2 stromal cells were
removed by incubation with collagenase, and then the remaining
osteoclasts were harvested for RNA as previously described (35).
mRNA was prepared using the poly(A) quick mRNA isolation kit (Stratagene).
-32P]ATP
using T4 polynucleotide kinase. The primer extension products were
separated in a 7% polyacrylamide sequencing gel together with a
sequencing reaction of the mCTR genomic clone 18.26 using the same
primer used in the primer extension reaction.
-32P]CTP utilizing T3 RNA polymerase. Total RNA from
mouse brain (200 µg) and mRNA from MDCT209 cells (20 µg) were
used as templates for RPA using 2 × 105 cpm of the
riboprobe following the manufacturer's protocol (Ambion). In addition,
mCTR cRNA sense strand derived from the same RPA construct was used as
a positive control that would yield a 376-bp protected fragment. The
RPA protected products were separated in a 5% sequencing gel and
analyzed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
-32P]ATP-end-labeled oligonucleotide probes located
internal to the expected PCR products for each experiment, such as mPE4
and E3b-R. The mCTR positive 5'-RACE products were subcloned into the
TA 2.1 vector (Invitrogen). The resultant cDNAs were characterized by sequencing from both orientations using T7 and M13-reverse primers.
-32P]ATP end-labeled
oligonucleotide probes E2a-F2, E2b-R, E2c-F, E3a-R, E3b'-F, and
E3b-R.
1253 relative to E2a) to the EcoRI site at the end of E2c
(+398) (
1253P2Bam-F and
1253P2Bam-R). The initial 859-bp P3 genomic
region (starting at
797 relative to E3b') was synthesized by PCR
using primers E3a-F and E3b'-R (
797P3ab'-F and
797P3ab'-R). The
identity and orientation of each construct was verified by sequencing.
In addition, three P2 deletion constructs were generated using the
KpnI, SacI, and NheI sites in the 5'
polylinker region and in the P2 promoter DNA (
806P2Kpn-F,
285P2Sac-F,
179P2Nhe-F, respectively) to drop successively larger
fragments after recircularizing the construct. Further P2 deletions
were derived from the
179P2Nhe-F plasmid as follows. The
30P2Afl-F
and the
179/
27P2NAf-F plasmids were generated by dropping the
148-bp NheI/AflII and the 453-bp
AflII/HindIII fragments, respectively, blunting,
and recircularizing, while the
178/+16P2NNs-F was generated by
subcloning the 194-bp NheI/NspBII fragment into
SmaI-cut pGL3basic. The
797P3ab'-F construct was used to
generate P3 deletions to
319 and
94 by digesting the plasmid with
NheI plus XhoI, which cut in the polylinker,
isolating the mCTR fragment, and redigesting it with
BsrI or MslI. The BsrI/XhoI and MslI/XhoI mCTR fragments were then blunted
and cloned back into the SmaI site of pGL3basic to make
319P3Bsr-F and
94P3Msl-F, respectively.
G Berthold) luminometer. The
results represent at least three repeats of each experiment. Luciferase
activity was normalized by total protein concentration determined using Coomassie protein assay reagent from Pierce.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic structure of the mCTR gene.
A, depiction of 13 mCTR genomic phage inserts
(numbered thick lines) plus a portion
of the gene obtained by PCR between exons E6 and E7
(CD-PCR). Phage inserts 13 and 15 do not overlap, so the
intron between E3b' and E3b is >13.9 kb. The designations of the exons
(E#) reflect the numbering system used for the pCTR coding
exons (22). The filled boxes portray locations of
the mCTR exons, and the intron lengths (kb) are denoted
below each one. The novel exonic regions described in this
report are included. Note that IVS10, between exons E10 and E11, is
only 81 bp long, and consequently those two exons appear as one. The
two putative translation starts (small arrows
marked aa) in the mCTR cDNA are split between exons E3a
and E3b. The three RNA starts described in this report are also denoted
(large arrows). *, the exact position of the P1
RNA start is not yet known. Various restriction enzyme cut positions
are indicated (B, BamHI; Bg,
BglI; K, KpnI; P,
PstI; X, XhoI). The order of the
PstI sites marked with dots is not certain.
B, correspondence between mCTR exons and putative protein
domains. The exons of the mCTR cDNA published by Yamin et
al. (21) plus the additional exon E1 sequences defined in this
report are depicted in relation to the mCTR protein domains previously
defined (21) presented schematically in the cellular membrane. The
horizontal size of each protein domain represents the position and
length of the exon(s) encoding it. The shaded protein domain
at the N terminus is the additional 17 amino acids encoded when
translation starts in E3a. The extracellular and intracellular protein
domains are black, and the seven transmembrane domains are
white.
).
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Fig. 2.
Sequence of upstream mCTR genomic and exon
E1. A, the uppercase letters
denote the genomic sequences defined as cDNA sequences by either
5'-RACE or RPA (see Fig. 2B), and the
lowercase letters indicate genomic sequence not
found in mCTR cDNAs. *, +, and represent the upstream end of
5'-RACE products derived from brain, kidney, and osteoclast RNAs,
respectively. The boldface G denotes the first
nucleotide of the previously published mCTR cDNA
(GenBankTM accession number U18542) (21) and the
underlined C denotes the extra C found in both
our genomic and cDNA sequences. The underlined
ag is a putative 3' splice acceptor. Several reverse primers
used in primer extension and 5'-RACE experiments are denoted by
boxed sequences. B, identification of
the 5'-end of exon E1 by RPA. A schematic representation of the mCTR
probe used in the RPA is presented above the gel.
A 532-bp mCTR genomic DNA fragment spanning from a BsaBI
site to a BamHI site encompassing the 5'-end region and part
of exon E1 was blunted with Klenow and subcloned into a SmaI
site of pBKS vector, linearized with BamHI, and used as a
template for synthesizing a 635-bp riboprobe with
[
-32P]CTP utilizing T3 RNA polymerase. A protected
fragment of ~245 nt is apparent (solid arrow)
in the RPA of 20 µg of MDCT209 mRNA (lane
4) and 200 µg of mouse brain total RNA (lane
5). In addition, 0.01 ng of mCTR cRNA sense strand derived
from the same RPA construct was used as a positive control that would
yield a 376-bp protected fragment (lane 3). The
mCTR probe annealed to yeast tRNA and both digested (lane
1) and undigested (lane 2) are shown.
For size determination, the following samples were run: a 100-nt RNA
ladder (lanes L) and an actin probe provided with
the RPA kit from Ambion annealed with yeast tRNA and digested
(lane 6) or undigested (lane 7; expect 304 nt) or
annealed with 20 µg of MDCT209 mRNA and digested to produce a
250-nt product (lane 8).
Primers
-32P]CTP-labeled riboprobe, which was used to
analyze mouse brain total RNA and MDCT209 mRNA (Fig.
2B). The RPA product observed with both RNA samples was
about 245 nt (Fig. 2B, solid arrow), thereby indicating that the 5'-end of exon E1 is located ~143 bp
further upstream. The additional size of exon E1 revealed by the RPA is
too small to explain the primer extension results discussed earlier.
These data therefore indicate that there must be another exon upstream
of exon E1 and separated from it by an intron of unknown length. The
sequence in the region identified as the 5'-end of exon E1 (derived
from the RPA) is 5'-AAGGTG-3'. The presence of a possible splice
acceptor junction, aag/GTG, suggests that the 5'-end of E1 is precisely
140 bp upstream of the 5'-end of the most upstream 5'-RACE cDNA
clone (see Fig. 2A). Between the 5'-RACE and the RPA
results, exon E1 has been extended 512 bp upstream of the previously
published cDNA, and the full-length of exon E1 is 682 bp (including
the 1-bp insert within the 3'-half of E1 observed in our cDNAs and
genomic clones). The exact position of the putative promoter P1 is yet
to be determined.
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Fig. 3.
Novel mCTR cDNAs indicate the presence of
additional promoters P2 and P3 and demonstrate alternate splicing.
A, the sequence of the novel exons E2a and E2b identified
through 5'-RACE and of exon E2c, which contains many conflicts with the
same region in the GenBankTM accession number U18542 file.
The structures of the cDNAs found starting with E2a are depicted
schematically below the sequence. *, the 5'-ends
of E2a found through 5'-RACE. The splice consensus sequences at intron
ends found in exon E2b are in boldface type and
underlined. There is extensive homology between the mCTR
P2-E2abc region and similar regions in hCTR and pCTR (see Fig.
9A), and we have elected to call the mCTR nucleotide aligned
with the hCTR and pCTR start +1 (here and in Figs. 4, 8, and
9A). B, the sequence of the novel exon E3b'
identified through 5'-RACE. The structure of the cDNA found
starting with E3b' is depicted schematically below the
sequence.
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Fig. 4.
Genomic sequence containing promoter P2 and
exons E2a, E2b, and E2c. Shown is a schematic of the mCTR genomic
region containing exons E1 through E3b' with the sequenced region shown
as indicated below. Restriction sites used to generate 5'
deletions in the P2 mCTR-pGL3 reporters used in Fig. 8 are denoted:
A, AflII; K, KpnI;
N, NdeI; Ns, NspBII;
S, SacI. Note that exons E2a, E2b, and E2c are
contiguous in the genomic sequence. Several putative transcription
factor binding sites found by computer analysis, using Transfac (75)
and other transcription data bases, are underlined.
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Fig. 5.
Genomic sequence containing promoter P3 and
exons E3a and E3b'. Shown is a schematic of the mCTR genomic
region containing exons E1 through E3b' with the sequenced region shown
as indicated below. The primers used to generate by PCR a
genomic fragment containing the P3 promoter that was utilized to create
the P3ab'-pGL3 reporters used in Fig. 8 as well as the ATG in E3a are
boxed. Restriction sites used to generate 5' deletions in
the P3 mCTR-pGL3 reporters used in Fig. 8 are denoted as follows:
B, BsrI; M, MslI. Several
putative transcription factor binding sites found by computer analysis,
using Transfac (75) and other transcription data bases, are indicated.
The NFAT/AP1 composite elements were located on the basis of an
algorithm designed by Kel et al. (76).
Exon/intron junction sequences of the mCTR gene
E3a) isoform was most clearly observed in the osteoclast, it is very
weakly present in kidney and brain. Comparison of the relative amount
of mCTR PCR products generated from the 3 mCTR-producing tissues
observed in Fig. 6, A, C, and D,
indicates that the P1 promoter does not appear to be a major
contributor to mCTR transcripts in kidney. It should be noted that
exons E2a, E2b, and E3b' were not detected in cDNA products derived
from P1 transcripts. This suggests that these exons are always spliced
out of P1 mRNAs, thereby adding weight to the argument that exons
E2a and E3b' are only used to initiate mCTR transcripts P2 and P3,
respectively. The P2.1 cDNA, that includes both exons E2a and E3a,
but not E2b (
E2b, +E3a), seems to be the predominant P2 transcript
isoform observed in all three CTR-positive tissues (Figs. 6D
and 7). The P2.2 cDNA (
E2b,
E3a), although clearly observed in
the osteoclast, was very weakly detected in the kidney and not detected
in the brain. A fourth novel cDNA isoform, P2.4, derived from the
P2 transcript was detected most clearly by the E2b probe and includes E2b but not E3a (+E2b,
E3a). The two E2b-containing cDNAs, P2.3 (+E2b, +E3a) and P2.4, were observed in osteoclast and brain but not in
kidney. These RT-PCR products were also hybridized with an
oligonucleotide probe from exon E3b', and no bands were detected (not
shown). This indicates that exon E3b' is always spliced out of
P2 mRNAs. Usage of exon E3b' was only detected in the osteoclast RNA (Figs. 6E and 7), suggesting that the putative P3
promoter may be osteoclast-specific. These data demonstrate that the
three primary mCTR transcripts are both expressed in a tissue-specific manner and alternatively spliced within the 5'-UTR region to form seven
different cDNA structures in a tissue-specific manner (Fig. 7).
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Fig. 6.
RT-PCR analysis of the tissue specificity of
mCTR 5'-UTR structures. RNAs from mouse osteoclasts
(Oc), kidney (K), liver (L), and brain
(B) were subjected to RT-PCR with different sets of PCR
primers to detect either all mCTR mRNAs (primers MT5-F + MT7-R)
(A), GAPDH mRNA (B), P1-mCTR mRNAs
(primers E1-F + E4-R) (C), P2-mCTR mRNAs (primers E1-F2 + E4-R) (D), and P3-mCTR mRNAs (primers E3b'-F + E4-R) (E). The PCR products were analyzed on ethidium
bromide-stained gels (first panel) and then
further analyzed by Southern blotting with
[ -32P]ATP-end-labeled oligonucleotide probe E3b-R,
E3a-R, E2a-F2, E2b-R, E2c-R, or E3b'-R as indicated (the last is not
shown, since it was negative except for the osteoclast P3-mCTR PCR
product). D, the positions of the L and
B lanes had to be switched. Promoter-specific PCR
of the relevant mCTR cDNA plasmids (P1.1, P2.1, and P2.3) were used
to prepare control PCR products C1, C2, and C3, respectively. Also, for
all PCRs, a negative control containing H2O instead of
cDNA was run (
) but not always shown. The bright
band in the M1 marker (100-bp ladder from New England
Biolabs) is 500 bp, whereas the bright band in
the M2 marker (100-bp ladder from Life Technologies, Inc.) is 600 bp.
The expected sizes of the PCR products are as follows: P1.1, 560 bp; P2.1, 452 bp; P2.1, 440 bp; P2.2, 332 bp; P2.3, 622 bp; P2.4, 514 bp; P3.1, 185 bp.
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Fig. 7.
Summary schematic of the seven mCTR
mRNA 5'-UTR structures and their tissue specificity.
1253P2Bam-F) and reverse
(
1253P2Bam-R) orientations. The forward and reverse full-length mCTR
P3 constructs contained 797 bp of sequence upstream of E3b' (including
exon E3a) as well as most of E3b' (
797P3ab'-F and
797P3ab'-R).
Several 5' deletions of P2 and P3 were also constructed:
806P2Kpn-F,
285P2Sac-F, and
179P2Nhe-F as well as
319P3Bsr-F and
94P3Msl-F.
Consistent with the RT-PCR data indicating the lack of P3 transcripts
in kidney (Figs. 6 and 7), only the mCTR P2 promoter constructs
generated luciferase activity in the MDCT cells (Fig. 8A,
solid bars). Also consistent with the RT-PCR data
demonstrating the presence of both P2 and P3 transcripts in osteoclasts
(Figs. 6 and 7), the forward-oriented P3 promoter construct
(
797P3ab'-F) was as active as the forward-oriented full-length P2
construct (
1253P2Bam-F) in the HD-11EM cells (Fig. 8A,
hatched bars). None of the reverse constructs
were active in either cell type, indicating that although the P2 and P3
genomic regions are likely to contain some enhancer function, they are
contributing a key promoter function to the pGL3basic vector. Although
relative to the pGL3basic vector, the mCTR P2-promoter constructs were
~10-fold more active in the HD-11EM cells than in the MDCT cells, the
P2 promoter constructs of different lengths demonstrated the same
relative activity to each other in both cell lines. The truncation from
1253 to
806 approximately doubled the P2 promoter activity, and the
truncation from
806 to either
285 or
179 once again doubled the
activity. In contrast, a truncation of the P3 promoter from
797 to
319 did not change the P3 promoter activity in either cell type (Fig.
8A), while further deletion to
94 halved the activity.
While the P3 region between
319 and
94 is clearly important for
full activity, it cannot act as a promoter-independent enhancer when
added to the c-Fos promoter (not shown).
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Fig. 8.
Transient transfections demonstrate that the
P2 promoter is transcriptionally active in both kidney and
osteoclast-like cells; however, the P3 promoter is only
transcriptionally active in osteoclast-like cells. A,
schematics of the mCTR E1-E3b' genomic (not to scale) along with each
of the constructs used to transiently transfect the MDCT murine kidney
cell line (black bars) and the HD-11EM chicken
osteoclast-like cell line (hatched bars). The
luciferase activity for each transfection was normalized for protein.
Transfections were done in triplicate within an experiment, and the
mean luciferase units/µg of protein for each construct was normalized
to the pGL3basic construct to yield the -fold activation. Each
bar is the mean of the relative luciferase activity from at
least three experiments ± S.D. The 94P3Msl-F construct was the
only one not transfected into the MDCT cells. B, schematics
of the expanded mCTR P2-E2abc genomic in the
179P2Nhe-F plasmid along
with each of the deletions derived from it that were used to
transiently transfect the MDCT murine kidney cell line
(black bars) and the HD-11EM chicken
osteoclast-like cell line (hatched bars).
Transfections were done as above, but the data are represented as a
percentage of the
179P2Nhe-F plasmid activity. Various restriction
enzyme cut positions are indicated (A, AflII;
B, BamHI; Bs, BsrI;
E, EcoRI; K, KpnI;
M, MslI; N, NheI;
Ns, NspBII; S, SacI).
179/+398P2Nhe-F) to generate
30/+398P2Afl-F,
179/
27P2NAf-F, and
178/+16P2NNs-F (Fig.
8B). Transfections into both cell types yielded similar results. Deletion from the 5' side to
30 (
30/+398P2Afl-F) and deletion from the 3' side to
27 (
179/
27P2NAf-F) yielded
constructs with very little activity. However, in both cell types, the
construct with a 3' deletion to +16 (
178/+16P2NNs-F) retained about
half of the activity as compared with the
179/+398P2Nhe-F construct. Therefore, while there may be some positive regulatory elements between
+16 and +398, the region between
179 and +16 is required for
promoter activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 region, an Inr element (YYANWYY)
at +1 (49), a DPE site (RGWCGTG) downstream near +30 (50, 51), and a
BRE site (SSRCGCC) at approximately
38 relative to the start of
transcription (52). Both promoters possess a possible YY1 binding site
(VKHCATNWB) at the putative transcription start that could be involved
in recruiting TFIIB (53, 54). The mCTR P2 promoter between the NheI site (
179) and the transcription start is relatively
GC-rich (70% GC) and contains 18 CpG (which represents 10.1 CpG/100 bp) and 21 GpC to give a ratio of CpG/GpC = 0.86. These
ratios are the hallmark of a CpG island (55). Promoters containing CpG islands have been proposed to be associated with replication origins and with transcriptional activity during embryogenesis (56). On the
other hand, the P3 promoter region between E3a and E3b' is only 36%
GC, and the
319 region is only 41% GC.
179 mCTR P2 region with the pCTR promoter sequence upstream from
"exon 1" (22) and a region of hCTR upstream of exons 2a-2c
(1392-1360, 1359-1175, and 1174-957, respectively, in human BAC
GS1-438P6; GenBankTM accession number AC005024) revealed a
high degree of homology and conserved sequence motifs for several
transcription factors (Fig.
9A). The homology between the
three species was very high in pairwise comparisons (~70%) for more
than 2 kb further upstream (not shown). Typical for many TATA-less
myeloid promoters, the proximal P2 promoter regions of the CTRs of all
three species contain two putative Sp1 sites (60), although their
positions are not identical. Among the more intriguing putative binding sites in the
179 P2 promoter (indicated in Figs. 4 and 9A)
are consensus sequences for several transcription factors known to be
important for myeloid gene expression: Spi-1/PU.1, myeloid zinc
finger-1 (MZF-1), and MBF, which overlaps a good consensus half-site
for either AP1 or CREB (for a review, see Ref. 61). Fitting with the
location of the P2 promoter within a CpG island, Jagger et
al. (30) found that a 2.1-kb fragment of the pCTR promoter that
resembles mCTR P2 was only able to direct expression of the
lacZ reporter in several embryonic and fetal tissues
that express mCTR but not in the adult kidney or bone of the transgenic mice. Similarly, among the more intriguing putative binding sites (indicated in Figs. 5 and 9B) in the
319 P3 promoter are
putative sites for NFAT + AP1, E-box binding proteins, STATs (GAS
sites), MZF-1, GATA, and Spi-1/PU.1. Comparison of the mCTR P3 region with the human BAC GS1-117O10 (GenBankTM accession
number AC003078), which contains part of hCTR, revealed a region of
high homology (~73%) between 172939 and 171751 (Fig. 9B,
only the
319 E3b' region is shown). Using 5'-RACE on RNA from human
osteoclasts, Nishikawa et al. (59) identified a 288-bp osteoclast-specific exon (located at positions 168711-168422 in GenBankTM accession number AC003078) spliced to exon E3.
This region lies between the P3 homology region and E3. Interestingly,
unlike the mCTR E3a (which is upstream of P3), the hCTR variably
spliced exon (71-bp insert) containing the upstream ATG is located
downstream of both the P3 homology region and the osteoclast-specific
exon identified by Nishikawa et al. (59).
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Fig. 9.
Comparison between the murine, human,
and porcine CTR promoter regions. A, three-way
comparison of the P2 promoter region between mCTR, hCTR, and pCTR. The
179 NheI P2 mCTR promoter region (GenBankTM
accession number AF333472) was aligned with hCTR (1578-1343 in
GenBankTM accession number AC005024, which was located
using the hCTR E2ac cDNA described by Nishikawa et al.
(59) (Gen- BankTM accession number AB022177) as the query
sequence with BLAST) and pCTR 2231-2460 E1 genomic region
(GenBankTM accession number Z31356) (22) using MACAW. The
+1 denotes the reported 5'-ends for the hCTR and pCTR and
the putative assignment for mCTR. Boxed regions
indicate regions of identity between all three species. B,
homology between the mCTR P3 promoter region and a region of hCTR. The
319 P3 promoter region of mCTR (GenBankTM accession
number AF333473) was aligned with the hCTR homology region
(Gen- BankTM accession number AC003078) found using mCTR
P3 as the query sequence in BLAST. Boxed regions
indicate regions of identity. In both A and B,
the putative transcription factor binding sites in mCTR denoted in
Figs. 4 and 5 are marked above the appropriate sequences.
Additionally, in B, the NFAT/AP1 sites found in hCTR using
the algorithm designed by Kel et al. (76) are marked
below the sequence by thick bars (none
were found in the P2 region shown in A). Various restriction
enzyme cut positions are indicated (A, AflII;
Bs, BsrI; M, MslI;
N, NheI; Ns, NspBII).
3, which makes them poor
translation candidates, although one such upstream open reading frame
is 51 amino acids long (AUG at +252 in E1). One open reading frame
(which encodes 14 amino acids) has an AUG (at +248 in E1) in a good
Kozak context for translation. The occurrence of upstream AUG codons
nearly always reduces the efficiency of initiation from downstream AUGs
(64). The P2 5'-UTRs have only one upstream open reading frame with its
AUG in a poor Kozak context, range in size from 249 to 487 nt, and are
all GC-rich (55-60% GC). The P3 5'-UTR has no upstream AUGs, is only
93 nt long, and is slightly AT-rich (48% GC). One possible purpose of
the generation of multiple mCTR 5'-UTRs is that mRNAs with
different untranslated exons can differ in their stability and
translational potential. When the translatability of mRNAs from the
same gene with both a long and a short 5'-UTR has been compared, the
short 5'-UTR usually is more efficiently translated (65). Indeed, in
some instances, the effect of the 5'-UTR on translation can be so
profound that a minor transcript from certain genes appears to be the
major functional mRNA (66, 67). It is therefore possible that
translation of mCTR protein from mRNAs with a short 5'-UTR is more
efficient than from mRNAs with a long 5'-UTR. The GC-rich
untranslated regions could have mRNA secondary structure that may
interfere with the translational process (68-70). It has been shown
that deletion of GC-rich untranslated sequences improves translation
efficiency of the guanylate cyclase gene (71) and the fibroblast growth factor-related oncoprotein INT-2 (72). Differences in the length of the
5'-UTR might also affect the rate of mRNA degradation. For
instance, platelet-derived growth factor B/c-sis
mRNA possessing a truncated 5'-UTR was more resistant to
degradation in response to cycloheximide and anisomysin than the
mRNA with a long 5'-UTR (73). Additionally, the various 5'-UTRs
could differentially affect mRNA compartmentalization, targeting
mRNA to be either translated immediately or stored for later use
(74). Therefore, it is possible that translation of the seven mCTR
cDNAs is differentially regulated, and the relative abundance of a
particular mRNA isoform may not correlate with its contribution to
the translated product.
3 integrin, acid
phosphatase, and CTR. These genes encode protein products that confer
upon the osteoclast the unique functional activities required for
attachment and resorption of the mineralized bone matrix. The CTR gene
appears to be induced during the terminal stages of osteoclast
differentiation coincident with the acquisition of bone resorbing
capacity (35). Characterization of molecular regulation of the CTR gene
in osteoclasts could lead to novel approaches in treating osteoporosis,
periodontal disease, inflammatory arthritis, and related bone disorders.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Chuxia Deng for the genomic library, to Dr. Peter A. Friedman for the MDCT cell line, and to Dr. Peter V. Hauschka for the HD-11EM cell line. We thank Dr. Philip E. Auron for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR45421 (to D. L. G.) and DK46773 (to S. R. G.).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) AF333472 (mCTR E1/E2 genomic), AF333473 (mCTR E3a/E3b' genomic), AF333474 (mCTR E3b genomic), AF333475 (mCTR E4 genomic), AF333476 (mCTR E5 genomic), AF333477 (mCTR E6 genomic), AF333478 (mCTR E7 genomic), AF333479 (mCTR E8a genomic), AF333480 (mCTR E8b genomic), AF333481 (mCTR E9 genomic), AF333482 (mCTR E10/E11 genomic), AF333483 (mCTR E12 genomic), AF333484 (mCTR E13 genomic), AF333485 (mCTR E14 genomic).
¶ Present address: Dept. of Conservative Dentistry, Faculty of Dentistry, Prince of Songkla University, Hatyai, Songkla, Thailand 90112.
** Recipient of an Arthritis Foundation Fellowship Award.
Present address: IDEC Pharmceuticals, Molecular Biology
Department, 11011 Torreyana Rd., San Diego, CA 92121.
§§ To whom correspondence should be addressed: Deborah L. Galson, Beth Israel Deaconess Medical Center, New England Baptist Bone and Joint Institute, Harvard Institutes of Medicine, Rm. 247, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0743; Fax: 617-975-5299; E-mail: dgalson@caregroup.harvard.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M007104200
2 The following sequences deposited into GenBankTM are referred to in this paper: the original mCTR cDNA sequence under GenBankTM accession number U18542 (21); human BAC GS1-438P6 under GenBankTM accession number AC005024; human BAC GS1-117O10 under GenBankTM accession number AC003078; hCTR E2ac cDNA under GenBankTM accession number AB022177 (59); and pCTR genomic under GenBankTM accession number Z31356 (22).
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ABBREVIATIONS |
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The abbreviations used are: CTR, calcitonin receptor; hCTR, human CTR; pCTR, porcine CTR; mCTR, murine CTR; UTR, untranslated region; STAT, signal transducer and activator of transcription; kb, kilobases(s); bp, base pair(s); nt, nucleotide(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RPA, RNase protection assay; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT, reverse transcription; AP1 and AP2, adapter primer 1 and 2, respectively; BAC, bacterial artificial clone.
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