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Endocrine Unit (H.J., B.L., F.Q., A.A.-S.), Massachusetts General Hospital, Boston, Massachusetts 02114; and the Netherlands Institute for Developmental Biology (M.K., L.D.), Utrecht, Netherlands
Address all correspondence and requests for reprints to: Abdul Abou-Samra, M.D., Ph.D., Endocrine Unit/Bulfinch 3, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114. E-mail: Samra{at}helix.mgh.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1–34), PTH(1–84) and hPTHrP(1–36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently isolated overlapping genomic clones encoding the human, mouse, and rat PTH/PTHrP receptor gene (10). The PTH/PTHrP receptor gene has several unique features that distinguishes it from genes of most G protein-coupled receptors. These features include a complex organization with 14 exons encoding the putative receptor protein and a 3' region that does not contain a typical polyadenylation signal (10). The PTH/PTHrP receptor gene was thought to be transcribed from a transcription start site lying upstream of the most 5' noncoding exon previously characterized (exon U) (11). In this report, we show that the PTH/PTHrP receptor gene uses alternate transcription start sites in a tissue-specific manner that results in several alternatively spliced transcripts with unique tissue distribution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Riboprobe synthesis
Genomic and cDNA sequences of interest were cloned into pGEM-7
or pcDNA1 vectors, linearized, and either T7 or SP6 RNA Polymerase
(Promega, Madison, WI) was used for antisense riboprobe synthesis
according to manufacturer’s recommendations.
Ribonuclease protection assay
The riboprobe (2.5 x 105 cpm) and total RNA
(10 µg) were coprecipitated with ethanol and 0.4 M
ammonium acetate. The pellet was resuspended in 30 µl hybridization
buffer (80% deionized formamide, 40 mM PIPES, pH 6.4, 0.4
M sodium acetate, 1 mM EDTA); incubated at 85 C
for 10 min, then at 45 C for 16 h; 300 µl RNAse digestion buffer
(10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200
mM sodium acetate) and 10 U RNAse ONE (Promega) were added
to each sample and incubated for an appropriate time (15 to 60 min) at
22 C; then 5 µl of a stop solution (10% SDS, 4 µg/µl transfer
RNA) and 825 µl 100% ethanol were added for precipitation; the
pellet was resuspended in 6 µl loading dye (80% deionized formamide,
10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol,
0.1% SDS); 3 µl of the sample was loaded onto an 8%
polyacrylamide/7 M urea gel and analyzed by
autoradiography.
Sequencing
Double-stranded DNA sequencing was performed using Sequenase
version 2.0 sequencing kit (United States Biochemical Corp., Cleveland,
OH).
Primer extension analysis
Ten picomoles of primer was end-labeled with 8 U T4
Polynucleotide Kinase (USB); 0.5 pmol of labeled primer and 30 µg
total RNA were combined in AMV primer extension buffer (50
mM Tris-HCl, pH 8.3 at 42 C, 50 mM KCl, 10
mM MgCl2, 10 mM dithiothreitol, 1
mM each deoxynucleotide triphosphate, 0.5 mM
spermidine) to 11 µl final volume; primer and RNA were denatured by
heating at 85 C for 10 min, then annealed at 52 C for 30 min, and
allowed to cool to room temperature for 10 min; 5 µl AMV PE 2x
buffer, 1.4 µl of 40 mM sodium pyrophosphate, 1 U AMV
Reverse Transcriptase (Promega), and 2.6 µl nuclease-free
H2O were added to each sample and allowed to incubate at 42
C for 45 min; the samples were ethanol precipitated and resuspended in
8 µl loading dye (98% deionized formamide, 10 mM EDTA,
0.1% xylene cyanol, 0.1% bromophenol blue) heated at 90 C for 10 min,
and 2 µl loaded onto an 8% polyacrylamide gel/7 M urea
gel.
RT-PCR
Ten micrograms of kidney total RNA was reverse transcribed using
10 U AMV-RT and 100 pmol of an antisense exon E1-specific primer (E1b);
PCR amplification was carried out using 100 pmol of a second antisense
exon-specific primer (Sb, 5'-) nested to the first and a sense exon
U3-specific primer (H2f, 5'-AAGGATCCGACCTGCTCAGGCCTGAA-3'). A band of
300 bp was amplified, cloned into pcDNA1, and sequenced.
Rapid amplification of cDNA ends (RACE) and cloning of the type III
splice-variant
RACE was carried out as described (12). Poly A+ RNA
(1 µg), isolated from rat kidney, was reverse transcribed in a final
volume of 20 µl at 42 C for 2 h in a buffer containing 10 U
ribonuclease inhibitor (RNasin), 1 mM dithiothreitol, 1
mM deoxynucleotide triphosphates, 10 U AMV reverse
transcriptase, 1x reverse transcriptase buffer (Promega), and 50 pmol
of primer R-2 (5'-AGGCTGTGCAAGTACAGCCC-3'), an antisense primer that
primes on exon M3 that encodes the third transmembrane domain. The
first-strand cDNA pool was diluted to 100 µl and run through a
Bio-Spin 30 spin column (Bio-Rad) to remove excess primer. Poly(dA)
tailing of the first-strand cDNA was carried out in 250 nM
dATP, 1x terminal deoxynucleotidyl transferase buffer, and 10 U
terminal deoxynucleotidyl transferase (Promega) at 37 C for 5 min. The
tailed cDNA was amplified by PCR using Taq polymerase
(Perkin-Elmer), 50 pmol of HSJ primer
(5'-AAGGATCCGTCGAC-ATCGATAATACGACTCACTATAAGGGAT17-3')
and 50 pmol of R-32 primer (5'-GGAGGCGAGAGACATGGAG-3'), an antisense
primer corresponding to the exon (M1) encoding the first transmembrane
domain. The resulting product was a smear that ranged between
approximately 0.3 to 1.3 kb. The product was digested with
BamHI, a restriction site included in HSJ primer, and Esp3I,
a site present in the exon (G) that encodes all potential glycosylation
sites, and ligated into a plasmid containing the full-length type I
cDNA with the 5' portion excised with BamHI and
Esp3I. The resultant DNA encoding exons U3 and U2 spliced to
E1 was characterized by sequencing.
Expression of PTH receptor splice variants in COS-7 cells
The new cDNAs were cloned in the expression vector, pcDNA1. The
cDNA constructs were transiently expressed in COS-7 cells using the
diethylaminoethyl-dextran transfection method. Cell surface expression
of the PTH receptor protein was determined using a double antibody
binding assay (13). The first antibody is directed against a synthetic
peptide that was based on the sequences encoded by the exon E2 region
(10). This antibody, G48, is specific for the rat PTH/PTHrP receptor.
Expression of functional PTH/PTHrP receptors was assessed by the
ability of COS-7 cells to bind
125I-PTHrP(1–36)NH2 (125I-PTHrP)
and to increase their intracellular cAMP accumulation after challenge
with [Nle8,18,
Tyr34]bPTH(1–34)NH2 (NlePTH), hPTH(1–84) and
PTHrP(1–36) (10-6 M) (13).
Immunofluorescence was performed using fluorescein (FITC)-labeled second antibody. Intact and NP40-permeabilized (0.1% NP40, 5 min on ice) were incubated with first antibody on ice for 2 h in PBS containing 5% FBS. The first antibody was removed, the cells were rinsed three times with PBS, and then incubated with FITC-labeled second antibody for additional 2 h. The cells were rinsed three times, fixed with 2% paraformaldehyde in PBS, and examined under a fluorescent microscope (13).
For Western blot COS-7 cells expressing type I or type III, transcripts were lysed with the SDS sample buffer and analyzed on 5–20% SDS-polyacrylamide gel. The gel was blotted on Immobilon-P filters (Millipore, Burlington, MA); the filters were blocked with 5% nonfat dry milk in PBS and incubated with the first antibody at a 1:2000 dilution followed by a peroxidase labeled antisheep second antibody. Chemoluminescence was used for detection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize the genomic sequences representing the new putative
exons, we used a riboprobe containing the longest new 5' sequences
(19H) to screen a rat genomic library. A new genomic clone (RG5, Fig. 1
) that overlaps with the previously described phages
(RG1, RG2, RG3 and RG4) (10) was obtained and subcloned in a plasmid
vector. Sequence analysis revealed that the new cDNA sequences of 19H
map to two 5' regions on the gene, exons U3 and
U2, that lie 2.5 and 4.8 kb, respectively, upstream of exon
S (Fig. 1). The previously identified untranslated exon is henceforward
called exon U1. The 3' end of exon U3 (EXON
U3 intron:CAG gtaagt) has a perfect consensus for splicing. The 5'
end (intron EXONU2:cag ATG) and the 3' end (EXON
U2 intron:TTG gtaagt) of exon U2 contain perfect splicing consensus
in their intronic regions; however, they have 1 and 2 bp mismatches,
respectively, with the splicing consensus in their exonic regions.
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), which has
the following splicing pattern: U3 to U2 to S
(skipping U1, Fig. 1
), and porcine kidney (16) (Fig. 1) which has the
following splicing pattern: U3 to S (skipping U2 and U1,
Fig. 1). Therefore, we hypothesized that rat kidney might have similar
splicing patterns. Primers from exons U3 and S were used to
force-clone a product containing U3 and S from rat kidney
cDNA that was reverse transcribed using the primer extension method
with a cDNA specific primer from exon E2. A PCR product was
obtained with a splicing pattern of U3 to U2 to
S, but no U3 to S was obtained. Thus, four splice variants
were identified: types I, II, III, and IV (Fig. 1).
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). Double antibody binding
assay to intact cells was used to quantitate cell surface expression of
the type III receptor protein (13). This assay makes use of a specific
antirat PTH/PTHrP receptor antibody (G48), which was raised against a
synthetic epitope from the sequences of exon E2, and an
125I-labeled second antibody. Compared to the expression of
the type I splice variant (R15B) the type III splice variant had low
cell surface expression that was 7% of that of type I (Fig. 3B).
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). However, despite the lack of
any detectable ligand binding by RRA, NlePTH, PTHrP(1–36), and
hPTH(1–84) caused a significant increase in cAMP accumulation in COS-7
cells transfected with the type III splice variant (Fig. 3C). Neither
PTH(3–34) or PTH(7–34) caused any significant stimulation of cAMP
accumulation (Fig. 3C). Additionally, PTH(7–34) blocked the effects of
NlePTH and PTHrP(1–36) on cAMP accumulation (Fig. 3D). Maximal
stimulation with NlePTH, PTHrP(1–36), and hPTH(1–84) was about
15–20% of that obtained in COS-7 cells expressing the type I splice
variant (Fig. 3E). Despite the low maximal stimulation, the
EC50 of NlePTH and PTHrP(1–36) for stimulating cAMP
accumulation in COS-7 cells expressing the type III splice variant was
not different from that observed in COS-7 cells expressing the type I
splice variant (Fig. 3E). Because surface expression, as measured by
antibody binding, was reduced to 7% of the type I variant, it is
likely that decreased maximal cAMP stimulation is secondary to
decreased expression. These data show that the type III splice variant
can transduce a cAMP signal when challenged by PTH and PTHrP ligands
and indicate that low levels of type III receptor are expressed on the
cell surface.
Intracellular localization of the type III splice variant
Because the type III splice variant does not have a typical signal
peptide, its expression may be mostly intracellular and/or it may be
improperly folded. Therefore, we used FITC-labeled second antibody to
study cellular expression of the type III variant. A faint
fluorescence, scattered over the cell membrane, was detected in intact
COS-7 cells expressing the type III splice variant (Fig. 4A); this contrasted with the intense immunofluorescence
of COS-7 cell membranes expressing the type I splice variant (Fig. 4B).
Permeabilization of the cell membrane with NP40 (0.1%) revealed an
intracellular immunoreactivity (Fig. 4C); this suggested that most of
the receptor protein of the splice III variant does not reach the cell
surface. Alternatively, because it lacks the signal peptide, the amino
terminus of the type III splice variant might be oriented toward the
cytoplasmic side instead of facing the extracellular side, or this
receptor may cross the plasma membrane 6 (or less) times instead of 7.
Therefore, we epitope-tagged the receptor tail with a 10 amino acid
epitope from c-myc, which is recognized by the monoclonal
antibody 9E10, and studied cell surface expression of the
epitope-tagged type III splice variant using both the monoclonal
antibody 9E10 and an anti-E2 peptide anti-PTH/PTHrP receptor antibody
(17). No immunostaining was detected with the 9E10 monoclonal antibody
in intact cells. After permeabilization, an abundant immunostaining,
similar to that shown in Fig. 4C, was observed.
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). In contrast, a 90-kDa broad band was
detected in COS-7 cells expressing the type I cDNA (Fig. 5). Because
the predicted size of unprocessed type I and III proteins is 60 kDa,
the 90-kDa broad band seen in COS-7 cells expressing the type I cDNA
suggests that the type I protein is glycosylated. Conversely, the
60-kDa compact band seen in COS-7 cells expressing the type III cDNA
suggests that the type III protein was not processed. To confirm this
hypothesis, we compared the receptor size in COS-7 cells expressing the
type I and type III splice variants in presence and absence of
tunicamycin which blocks glycosylation. Treatment of COS-7 cells with
tunicamycin decreased the type I receptor size from 90 kDa to 60 kDa
(Fig. 5). Conversely, tunicamycin treatment did not influence the size
of the type III receptor (Fig. 5). Additionally, the size of the
nonglycosylated band of the type I protein was slightly smaller than
that of the type III protein (Fig. 5). These data indicate that the
type III splicing results in a receptor that is expressed poorly on the
cell surface, which is not glycosylated and does not have a functional
signal peptide.
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). Three major bands
were protected with the riboprobe containing exons U3, U2, and E1.
These bands represent the following transcripts: type III (U3 + U2 +
E1 = 274 bp), types I and type II [exon E1 only = 103
nucleotides (nt)] and type II (exons U3 + U2 = 171 bp); the
abundance of the type III transcript is about 5–10% of the total
transcripts. A fourth less abundant 114-nt band was protected with type
III riboprobe (Fig. 6A). This band may represent exon U3 only, which
corresponds to the type IV transcript recently cloned from porcine
kidney (16). Complementary data were obtained using a riboprobe
containing exons S and E1. With the latter riboprobe, three bands were
specifically protected: a 223-nt band corresponding to exons S and E1,
a 120-nt band corresponding to exon S, and a 103-nt band corresponding
to exon E1. The 223-nt band represents both type I, type II, and type
IV transcripts (exons S + E1 = 223 nt) and the 103-nt band
represents the type III transcript (exon E1 = 103 bp). A 120-nt
band that has the size of exon S was also protected (Fig. 6A). This
band may represent another splice variant in which exon S is not
spliced to exon E1.
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Heterogeneity of the size of the PTH/PTHrP receptor transcript,
observed by Northern blot of total and poly(A+) RNA from
rat kidney, was reported (18). The main transcript was estimated to be
about 2.5 kb, whereas other smaller and larger but less abundant
transcript were observed in rat kidney (18). To test whether exons U3
and U2 belong to a different size transcript we used cDNA fragments
representing exons U3 and U2 and exons S and E1 to probe
poly(A+) RNA prepared from rat and mouse kidney (Fig. 6B).
Both probes hybridized to similar size transcripts (
2.5 kb); this
suggested that the heterogeneity of the size of the PTH/PTHrP receptor
transcripts seen on the Northern blot is not due to the alternate
promoter usage and/or alternative splicing of the gene in its 5'
region.
Tissue distribution of the PTH/PTHrP receptor splice variants
To study tissue distribution of the different splice variants of
the PTH/PTHrP receptor transcript, we performed RNAse protection assays
on total RNA prepared from rat kidney brain, heart, liver, lung, ovary,
placenta, skeletal muscles, testis, uterus, and ROS 17/2.8 cells (Fig. 7). The data show that kidney contains the most abundant
expression of the PTH/PTHrP receptor transcripts and that transcripts
starting at U3 are limited to kidney (types II, III, and IV, Fig. 7) and ovary (only type IV, Fig. 7). Conversely, all the
tissues that were positive for PTH receptor transcript showed a
protection of exon S or exons S and E1 using riboprobes containing
either exon S alone or exons S and E1 (not shown). These data indicate
that all the other tissues, except ovary, transcribe the PTH/PTHrP
receptor gene from a start site other than that of U3; this site is
likely to start at U1 as previously reported (11).
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) was used as a riboprobe. This
riboprobe protected a 90-bp band. This indicates that the transcription
start site that is used by the U1 promoter is 90-bp upstream of the
SstI restriction site (Fig. 8A).
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). The genomic riboprobe protected a band
in rat kidney RNA that was two base pairs longer than the band
protected in COS-7 cells RNA expressing 19H (Fig. 8B); this indicated
that the transcription start site is only 2 bp upstream of the cloned
19H cDNA. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The multi-exonic structure of the PTH/PTHrP receptor gene raised the possibility that novel PTH/PTHrP receptor variants may arise by alternative splicing of this complex gene (10). Several alternatively spliced transcripts may occur in different regions of this gene. This manuscript, however, focused solely on the 5' end of the PTH/PTHrP receptor gene and uncovered alternative usage of two promoters. The distal promoter results in the transcription of one single transcript that has a wide tissue distribution. The activity of the proximal promoter is restricted to kidney and ovary and results in three alternatively spliced transcripts.
The findings that the amino terminus and the carboxy-terminal tail of the type III splice variant are not accessible extracellularly and that abundant immunoreactivity could be detected intracellularly suggest that the type III splice variant is mainly expressed intracellularly and/or that the type III splice variant is misfolded in a way that both amino-terminal and carboxy-terminal ends are oriented intracellularly. The fact that the type III splice variant can transmit a cAMP signal across the plasma membrane with a specificity and EC50s that are similar to those of the type I splice variant suggest that the type III splice variant is appropriately folded within the plasma membrane. This conclusion is supported by the finding that certain regions within the amino-terminus of the receptor are required for activation of the receptor by PTH and PTHrP (14, 15) and by the experimental observation, using epitope-tagging, that the carboxy-terminal tail of the type III splice variant protein is located intracellularly. The low cell surface availability of the type III splice variant may be explained by deficiency in signal peptide cleavage and inefficient glycosylation.
The ability of the type III splice variant to transmit a low cAMP signal when challenged with PTH and PTHrP supports the hypothesis that this splice variant is minimally expressed on the cell surface. This hypothesis was substantiated by the observation that receptor immunoreactivity was minimally detectable on the cell surface, whereas receptor immunoreactivity was abundant intracellularly. Recently, Usdin et al. (22) has isolated a new cDNA encoding a receptor that is highly homologous to the PTH/PTHrP receptor. The novel receptor, named PTHR2, is activated by PTH but not by PTHrP. Usdin et al. compared the signaling properties of the PTH2R to those of another PCR product that is identical to the human PTH/PTHrP receptor (8, 14, 15) except that it lacks the sequence of the signal peptide (22); this PCR product is the human version of the rat type III splice variant described above. The molecular cloning of type III splice variants from two different species in two different laboratories, and the finding of a type III transcript in rat kidney total RNA using RNAse protection assay, indicate that the type III splicing pattern is not the product of a cloning artifact. Additionally, the human type III splice variant transmits a low cAMP signal when challenged with PTH or PTHrP (22); these data agree with ours and further support the conclusion that the type III splice variant is expressed on the cell surface only minimally.
Translation of the type I, II, and IV mRNAs is predicted to start from
the same AUG codon in exon S. In contrast, translation of the type III
splice variant initiates at a novel AUG codon upstream of exon E1. The
latter conclusion is based on the fact that an antibody against a
synthetic peptide from the sequences of exon E2 (see Materials
and Methods) reacted with the type III receptor expressed in COS-7
cells and that the only AUG codon that is in-frame of exon E1 is the
first codon of exon U2 (double underlined in Fig. 2). The
sequence in this region (CAGAUGAGG) is conserved in the
mouse, rat, and human genes. The other AUG codons in this region are
either not in-frame or are located downstream of exon E1. It has been
shown previously that when two in-frame AUG codons are present in an
mRNA molecule the first AUG codon (in this case the one in U2) is
favored by the ribosomal machinery (23). Therefore, although it does
not have the Kozak consensus, the only upstream AUG codon that could
read through the receptor sequence is the one in U2.
The absence of an efficient signal peptide resulted in an intracellular localization of the type III protein; this may increase the degradation of the type III protein. However, most G protein-coupled receptors do not have a signal peptide. Thus the signal peptide is not an absolute requirement for expression and cell-surface targeting of G protein-coupled receptors.
The surprising observation that certain splice variants are missing the hydrophobic signal sequence, usually required for the proper extracellular positioning of long amino terminal extracellular domains, suggests the possibilities that this alternate splice choice might serve a novel function. Our data that the type III splice variant protein is mainly found intracellularly raises the possibility that the hydrophilic domain that replaced the hydrophobic signal peptide may function to target the type III splice variant to an intracellular compartment. In that regard, it has been recently shown that the PTHrP molecule has a basic domain that functions as a nuclear targeting sequence in COS-7 cells to target the newly synthesized PTHrP toward the nucleus and that nuclear PTHrP immunoreactivity was detected in murine bone cells (24). We speculate that the type III splice variant may represent an intracellular PTHrP receptor for an intracellularly targeted PTHrP ligand. Alternatively, the type III splice variant may represent a regulated process for splicing of the gene toward a less active form of the receptor.
In conclusion, the data show that the PTH/PTHrP receptor gene uses alternate transcription start sites in a tissue-specific manner and is subject to alternative splicing in its 5' untranslated exons. These processes result in at least four different splice variants; three of which are translated from the same AUG codon in exon S which encodes the receptor’s signal peptide; the fourth splice variant is translated from a different AUG codon without having a hydrophobic signal peptide. The latter receptor protein is not well expressed on the cell surface, however; it could be stimulated by PTH and PTHrP to increase cAMP accumulation.
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Footnotes |
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Received October 10, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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