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Experimental Studies |
Department of Physiology, McGill University (J.D.B., M.Y.K., H.S.L., G.N.H., D.G., J.H.W.), Montreal, Quebec, Canada H3G 1Y6; the Department of Medicine and Calcium Research Laboratory, McGill University and Royal Victoria Hospital (M.M., G.N.H., D.G.), Montreal, Quebec, Canada H3A 1A1; and the Department of Pediatrics, Chiba University School of Medicine (M.M., T.Y.), Inohana, Chuo-ku, Chiba, Japan
Address all correspondence and requests for reprints to: Dr. J. H. White, Department of Physiology, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6. E-mail: jwhite{at}physio.mcgill.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PTHR belongs to the vast family of G protein-coupled receptors containing seven transmembrane domains. The organization of the PTHR gene shows that it is closely related to a subfamily of receptors for peptide hormones, including calcitonin, vasoactive intestinal peptide, glucagon, and GH-releasing peptide (10, 11, 12, 13, 14, 15). Expression of the mouse PTHR gene is controlled by at least two promoters, designated P1 and P2, which give rise to transcripts differing in their 5'-untranslated regions (5'UTRs) but not their coding sequences (11, 16). In mice, expression from P1 is restricted mainly to kidney, giving rise to transcripts containing 5'UTRs composed of two exons, U1 and U2, spliced to the signal sequence exon (16). Expression of P2, on the other hand, is not tissue specific and, therefore, is likely to be largely responsible for the broad expression pattern of the PTHR. P2-specific transcripts contain a single 5'-UTR exon, U3, spliced to the signal sequence exon (11). Recently, a second PTH receptor (PTH2) has been characterized, with only 20% homology to the PTHR. PTH2 receptor is expressed in the brain and, to a lesser extent, in pancreas, testes, and placenta, but not in kidney (17). This receptor appears to bind PTH with much higher affinity than PTHrP (18).
Several clinical syndromes characterized by end-organ resistance to PTH
have been described that are associated with hypocalcemia, high levels
of endogenous circulating PTH, and the absence of a normal increase in
urinary cAMP excretion after the administration of exogenous PTH (19).
These have been collectively termed pseudohypoparathyroidism (PHP). PHP
type 1a (PHP1a) is associated phenotypically with resistance to the
action of several hormones in addition to PTH and the presentation of
Albright’s hereditary osteodystrophy (AHO) (19, 20, 21). The stigmata of
PHP1a include obesity, short stature, round face, and brachydactyly.
PHP1a is often associated genotypically with one of a number of
heterozygous mutations in the gene encoding
Gs
, which disrupt its function. However,
apparent cases of PHP1a have been characterized in which no mutations
in Gs have been found (22, 23, 24), suggesting
that PHP1a can arise from more than one type of defect in PTH
signaling.
PHP1b is not associated with a reduction in Gs
expression or AHO. PHP1b patients show a defect in renal PTH signaling,
but an apparently normal response to PTH in bone (19), suggesting a
defect in either renal PTHR function or expression (25). Recent gene
ablation experiments indicate that severe skeletal malformations are a
consequence of disruption of receptor signaling, indicating that
mutations in the PTHR coding sequence are an unlikely site for the
defect in PHP1b (26). This has been confirmed by direct studies of
genomic DNA from patients with PHP1b reporting the absence of such
mutations (27, 28). On the other hand, the existence of multiple
promoters in the murine gene raised the possibility that if such
promoters are conserved in the human gene, mutations that disrupt
tissue-specific expression of the PTH gene may be responsible for the
syndrome. Two human PTHR (hPTHR) complementary DNAs (cDNAs) have been
isolated from kidney libraries that contain 5'UTRs similar to those
present in renal-specific transcripts in the mouse (29, 30), raising
the possibility that the mouse and human gene structures are conserved.
Thus, mutations in key elements controlling tissue-specific promoter
activity in one of the 5'-untranslated region exons resulting in
abnormal splicing, stability, or translation of the PTHR messenger
ribonucleic acid (mRNA) could account for a kidney-specific defect in
PTHR action. To test this hypothesis, we have cloned and characterized
the upstream region of the hPTHR gene and studied its expression in the
human kidney. In addition, PTHR genomic sequences from normal
individuals as well as from seven patients with PHP1b were cloned and
analyzed for potential alterations affecting either the expression or
function of PTHR gene transcripts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients with PHP1b included five Japanese and two Caucasians,
all of whom were sporadic cases. The diagnosis of PHP1b was established
by the following criteria: hypocalcemia, hyperphosphatemia, elevated
serum PTH levels (more than twice the upper limit of normal), normal
serum magnesium levels, normal renal function, absence of AHO, and
normal thyroid function. In addition, all patients showed minimal
urinary cAMP elevation after injection of exogenous hPTH-(1–34). Serum
PTH was determined using a commercially available two-site
radioimmunometric assay (Nichols Institute, San Juan Capistrano, CA) or
a carboxyl-terminal RIA with PTH-(1–84) as a standard and a PTH
(1–84) tracer (31). Characteristics of the patients are presented in
Table 1
. Lymphoblastoid cell lines derived from PHP1b
patients were established by Epstein-Barr virus transformation (32).
All individuals in this study provided informed consent.
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The probe used for screening was generated using four
overlapping primers (Fig. 1A). Primer sequences were
derived from the 5'UTR of a human kidney cDNA clone of the PTHR (29),
and are homologous to mouse exons U1 and U2. This strategy constructs a
148-bp hPTHR 5'UTR probe using a two-step PCR strategy. Sequences of
primers hPTHR1 (62-mer) and hPTHR2 (63-mer) overlap by 16 bp. Primers
hPTHR3 (33-mer) and hPTHR4 (33-mer), which overlap hPTHR1 and hPTHR2,
respectively, extend the homology with hPTHR 5'UTR sequences to 148 bp
and contain flanking EcoRI sites. The sequences of the
primers are as follows: hPTHR1,
5'-GTGGCCAACTTGAGTCTGCTCTGCAGCTTTAGGCCCGACTTGGAAGGCCCATGGGCTGCAGA-3';
hPTHR2,
5'-GTCCTGGACACTACCACTCTTCGGCTGTCTGGACCTCAGTTTCCTCATCTGCAGCCCATGGGC-3';
hPTHR3, 5'-AATGAATTCGCC-TCCCCGTGGCCAACTTGAGTC-3'; and hPTHR4,
5'-AATGAATT-CAGTTGTGTGTCCTGGACACTACCA-3'. PCR was performed as
follows. Five cycles (95 C, 1 min; 72 C, 30 s; 50 C, 1 min)
were performed with primers hPTHR1 and hPTHR2 using Vent polymerase
(New England Biolabs, Beverly, MA). One twentieth of the reaction was
used for a second round of PCR with primers hPTHR3 and hPTHR4 for an
additional 25 cycles under the same conditions as those described above
to yield a full-length PCR product, which was digested with
EcoRI and inserted into Bluescript SK+.
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One million independent clones of a 
DASH normal human
genomic DNA library (Stratagene, La Jolla, CA) were screened.
Prehybridization and hybridization were performed for 2 h and
overnight, respectively, at 42 C in 40% formamide, 6 x SSPE,
1 x Denhardt’s solution, 10% dextran sulfate, 1% SDS, 25
mmol/L sodium phosphate (pH 7.5), and 10 µg/mL denatured salmon sperm
DNA. The hybridization was performed in the presence of 106
cpm/mL probe labeled with 32P by random priming. Positive
clones were subjected to three rounds of plaque purification, and DNA
was extracted using SDS-chloroform extraction of liquid cultures. Two
positive clones were further characterized by Southern blot analysis
using synthetic oligonucleotide probes hPTHR1 or hPTHR4 (Fig. 1A
) or a
560-bp XhoI-ApaI restriction fragment containing
the mouse U3 exon. The two clones, designated A and B, contained
inserts of 18 and 22 kilobases (kb), respectively, which overlapped
over a length of 18 kb (Fig. 1B). The U1 region was subcloned as a
5.5-kb BamHI fragment, whereas U2, U3, and SS exons were
subcloned as a 3.6-kb BamHI fragment.
Southern blot analysis of PHP1b patient genomic DNA
DNA was extracted from whole blood and lymphoblastoid cell lines derived from PHP1b patients. Ten micrograms of genomic DNA were digested to completion with BamHI, KpnI, PstI, PvuII, and SacI restriction endonucleases under the conditions recommended by the supplier (Boehringer Mannheim, Indianapolis, IN), electrophoresed on 0.8% agarose gels, and transferred to Hybond-N+ membrane (Amersham, Arlington Heights, IL). Probes were labeled by random priming using a Ready-To-Go DNA labeling kit (Pharmacia, Piscataway, NJ). Hybridization was carried out at 65 or 68 C, depending on the probe used, for 18 h in a solution containing 5 x SSPE, 5 x Denhardt’s, 0.5% SDS, and 20 µg denatured salmon sperm DNA/mL. Filters were washed at room temperature with 2 x SSPE-0.1% SDS twice for 10 min at 65 or 68 C, with 1 x SSPE-0.1% SDS for 15 min, and at 65 or 68 C with 0.1 x SSPE-0.1% SDS four times for 15 min. Autoradiography was performed at -70 C on Kodak XAR-5 film (Eastman Kodak, Rochester, NY) using intensifying screens.
PCR amplification of PHP1b patient genomic DNA
PCR was performed using 10 ng DNA, 20 pmol of each primer, 1 x Promega Taq buffer [10 mmol/L Tris (pH 9.0) 50 mmol/L KCl, 0.1% Triton X-100, 2.5 mmol/L MgCl2] (Madison, WI), 200 mmol/L deoxy-NTPs, 1 mmol/L MgCl2, and 2 U Taq DNA polymerase (Promega) in a total volume of 20 µL. The primers for U1 were: forward, 5'-ACAGAATCCTGGGCATCTGAAACACC-3'; and reverse, 5'-GAATTCGTCTGTCTGCCCATAGCAC-3'. Samples were cycled at 94 C for 1 min, 50 C for 1 min, and 72 C for 30 s for 5 cycles, and then at 94 C for 30 s, 55 C for 1 min, and 72 C for 30 s for 30 cycles, followed by 1 cycle at 72 C for 10 min. For U2 sequences, the forward and reverse primers were 5'-CAGAATTCTTGGGCTTGACAGATTTGC-3' and 5'-ATACTGCAGAAACTGAGGCAGAGGGAC-3'. Samples were cycled at 94 C for 1 min, 52 C for 1 min, 72 C for 30 s for 5 cycles, and then at 94 C for 30 s, 57 C for 1 min, and 72 C for 30 s for 30 cycles, followed by 1 cycle at 72 C for 10 min. To amplify sequences containing U3, nested PCR was performed using 4 primers. The first round of PCR was performed at 0.75 mmol/L MgCl2, and samples were amplified at 95 C for 1 min, 52 C for 1 min, and 74 C for 30 s for 5 cycles, and then at 95 C for 30 s, 57 C for 1 min, and 74 C for 30 s for 35 cycles, followed by 1 cycle at 74 C for 10 min, using the forward and reverse primers, 5'-AAGAATTCGCCTCTAGCGCAATGTCCC-3' and 5'-CAATGGAT-CCGAGACAGAGCAGCCTGCTGCTC-3', respectively. The second round of PCR was performed on 1/10th of the first amplification product at a MgCl2 concentration of 1 mmol/L under cycling conditions identical to those described above, using forward and reverse primers (5'-AAGAATTCTCTCGGCCTCTCCACACTC-3' and 5'-CAATGGATCCGACTCCGGCCACTTCC-3', respectively). For the SS exon, the primers were: forward, 5'-ACGGAATTCAGCCTGACGCAAGCTCTGCACC-3'; and reverse, 5'-TTACGGATCCTGGATCAGAGGGGACTCTCAC-3'. Samples were cycled at 94 C for 1 min, 54 C for 1 min, and 72 C for 30 s for 5 cycles, and 94 C for 30 s, 58 C for 1 min, and 72 C for 30 s for 30 cycles, followed by 1 cycle at 72 C for 10 min.
Ribonuclease (RNase) protection analysis
Probes for RNase protection were synthesized by in vitro transcription of DNA fragments using 3 U T3 or T7 RNA polymerase; 0.5 mmol/L each of UTP, ATP, and GTP; 10 mmol/L CTP; 60 µmol/L dithiothreitol; 1 U RNAguard (Pharmacia); and 50 µCi 32P-labeled CTP in 1 x Promega transcription buffer (40 mmol/L Tris (pH 7.5) 6 mmol/L MgCl2, 2 mmol/L spermidine, 10 mmol/L NaCl) at 37 C for 1 h, and then treated with 100 U fast protein liquid chromatography pure deoxyribonuclease 1 (Life Technologies, Grand Island, NY) for 10 min at 37 C. The reaction was stopped by adding 80 µL 0.1% SDS, and the probe was purified on a Sephadex G-50 spin column. RNase protections were performed using 20 µg total human kidney RNA or yeast transfer RNA (tRNA; as a control). Samples were precipitated with 105 cpm [32P]CTP-labeled probe, resuspended in 30 µL 80% deionized formamide, 40 mmol/L piperazine-NN'-bis 2 ethane sulphoric acid (pH 6.4), 0.4 mol/L sodium acetate, and 1 mmol/L ethylenediamine tetraacetate; denatured for 5 min at 85 C; and incubated overnight at 50 C. Digestion was performed for 1 h using 3 U RNase 1 (Promega) according to the manufacturer’s instructions, and products were run on a 6% polyacrylamide-urea gel. Autoradiography was performed for 48 h at -70 C with two intensifying screens. The integrity of the probe was checked by running 250 cpm in parallel on the gel.
Primer extension analysis
Ten picomoles of primer were labeled for 1 h at 37 C with
[
-32P]ATP using T4 polynucleotide kinase. One tenth of
this reaction was incubated with 10 µg of either human kidney total
RNA or yeast tRNA as a control overnight at 55 C in 300 mmol/L KCl, 20
mmol/L Tris-HCl (pH 8.0), and 2 mmol/L ethylenediamine tetraacetate in
a final volume of 26 µL. Samples were then put on ice, and 4 µL 25
mmol/L Tris-HCl (pH 8.0), 60 mmol/L MgCl2, 10 mmol/L
dithiothreitol, 5 mmol/L deoxy-NTPs, 2 U RNAguard (Pharmacia), and 100
U Moloney murine leukemia virus reverse transcriptase (Life
Technologies) were added. After 90 min at 43 C, the enzymes were
inactivated for 10 min at 75 C, and the reaction was extracted once
with phenol and ethanol precipitated. One quarter of the reaction was
denatured for 2 min at 80 C and run on a 6% denaturing polyacrylamide
gel along with a sequencing reaction as a mol wt marker.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hPTHR cDNA clones have been isolated containing 5'UTR sequences
that correspond to mouse PTHR gene exons U1 and U2 (29). The human
sequences were used to design oligonucleotides that were assembled by
PCR to generate a 148-bp DNA fragment (Fig. 1A). This fragment was used
to probe a human genomic DNA library, resulting in the isolation of two
independent clones, A and B, which are identical except for
approximately 4 kb of additional sequence at the end of clone A (Fig. 1B). Sequences homologous to mouse UTR exons U1, U2, and U3 as well as
the exon SS containing the signal sequence were found by Southern
analysis (not shown) to be located on two contiguous BamHI
fragments of 5.5 and 3.6 kb, respectively (Fig. 1B). The exonic
structure of the 5'-end of the PTHR gene is well conserved between
mouse and human (Fig. 1B). Sequences corresponding to all three mouse
UTR exons are present in the human gene, although the U1 and U2 exons
in the human gene are more widely separated than their mouse
counterparts.
Mapping the P1 and P2 promoters of the hPTHR gene
We have used primer extension and RNase protection analyses of
human kidney RNA to map the sites of transcription initiation at the
5'-ends of human exons U1 and U3. Several human P1 promoter
transcription initiation sites were detected by RNase protection
analysis (Figs. 2B and 3). Several sites
were also found by primer extension that correspond closely to those
detected by RNase protection (Fig. 2C). The human P1 promoter is
similar to the mouse promoter (16) in that it is composed of several
initiation sites. Although the initiation sites are not identical in
the mouse and human sequences, they are similarly distributed, being
generally downstream of a conserved palindromic (A+T)-rich motif (Fig. 4A). It is noteworthy that the human P1 promoter contains a consensus
AP-1 site that is lacking in the mouse promoter (Fig. 4A).
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). A probe complementary to
72b of U3 lying between NcoI and SacI sites
protected a fragment of the expected size (Fig. 2B, lane 2). A probe
complementary to sequences upstream of the SacI site in U3
protected a 95b fragment (Fig. 2B, lane 5, and Fig. 3), placing the
initiation site for the human P2 promoter 15 bp downstream from that of
the mouse promoter (Fig. 4C). Consistent with this observation, a
primer extension product obtained using the U3UP primer was detected
which terminated at the same 3'-nucleotide (Figs. 2C and 3). Thus, like
their mouse counterparts, human P2 transcripts are initiated from a
single site. The proximal P2 promoter regions are very similar,
containing conserved Sp1 sites. Both promoters also contain consensus
sequences recognized by the ets class of transactivators
(Fig. 4C). The nucleotides around the mouse initiation site are not
conserved in the human promoter, which may account for the difference
in initiation sites. It is not surprising that the initiation sites of
the P1 and P2 promoters are not conserved between mouse and human,
given that neither promoter contains a TATA box to control start site
selection.
The sequencing and promoter mapping studies are summarized in Figs. 3 and 4. A 2.0-kb sequence of the U1 region, 2.4 kb of contiguous
sequence containing exons U2 and U3, and 400 bp containing SS are shown
in Fig. 3. Human U1 and U2 exon share 74% and 73% homology,
respectively, with the corresponding sequences in the mouse (Fig. 4, A
and B). The human U3 exon is 92% homologous to the mouse U3 sequence,
a degree of conservation that is remarkable considering that no human
cDNAs containing U3 sequence have been identified to date.
Southern blotting analysis of genomic DNA from PHP1b patients
The structure of genomic DNA from a normal individual and seven
patients diagnosed with PHP1b was compared by Southern blotting
analysis using four probes specific for the U1, U2, U3, and SS exons
(Fig. 1B), which encompassed 7 kb of genomic DNA. PvuII,
PstI, SacI, or BamHI digests of normal
DNA and DNA from four PHP1b patients that were analyzed with all four
probes (Fig. 5 and Table 2) generated
restriction patterns predicted from the map of the normal PTHR gene
(Fig. 1) and did not reveal any differences between the samples.
Similarly, analysis of KpnI digests of DNA from all seven
PHP1b patient samples with any of the four probes did not reveal any
differences from normal DNA (Table 2). These results suggest that there
are no gross rearrangements of genomic DNA in the promoter region of
the PTHR gene in the seven PHP1b patients tested.
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A finer analysis of genomic DNA encompassing exons U1, U2, U3, and
SS from all seven PHP1b patients and a normal control was performed by
PCR amplification using primers flanking each exon, indicated in Fig. 3. In all cases, fragments were amplified from patient DNAs that
appeared to be identical in length to the normal DNA control (not
shown) and to the size predicted from the cloned normal gene. Amplified
fragments corresponding to exons U1, U2, U3, and SS from all seven
patients and a normal subject were subcloned into Bluescript
SK+, and six recombinants of each insert were sequenced. No
changes in exonic DNA or spliced donor or acceptor sequences were found
in any of the clones tested, strongly suggesting that the structure or
splicing of the 5'UTRs of PTHR mRNAs is not affected in these PHP1b
patients. These analyses also included 100 and 80 bp of P1 and P2
promoter sequences, respectively. Again, no differences were found in
DNA sequences of PHP1b patients and those of normal subjects (not
shown). This suggests that the positions of transcriptional initiation
of the two promoters are probably not affected in PHP1b patients, given
that promoter-proximal elements control initiation site selection.
Taken together, the above results suggest that the structural integrity
of the 5'UTR PTHR mRNAs expressed from either P1 or P2 promoters is not
affected in the PHP1b patients tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both human and mouse P1 promoters contain initiation sites spread over at least 100 nucleotides. The striking common feature between the two promoters is a conserved 32-bp palindromic (A+T)-rich sequence located in the initiation region. Interestingly, the proximal region of the human promoter contains a consensus AP-1 site that is not found in the mouse, suggesting that the two promoters may be differentially regulated. It is noteworthy that promoters such as P1 with multiple start sites have the potential to produce transcripts with 5'UTRs with differing capacities to generate stem-loop structures. Such structures have been proposed to control the efficiency of translation of mRNAs (33). Our analyses of the 5'UTRs of different transcripts expressed from the human P1 promoter indicate that they can form stem-loop structures of differing stabilities, suggesting that they may be translated with varying efficiencies (data not shown).
The human and mouse P2 sequences are well conserved, and several Sp1 and ets factor-binding sites are present in both species. Unlike the P1 promoters, the P2 promoters direct expression from single initiation sites that differ by 15 bp in the mouse and human sequences. This shift may be due to the fact that nucleotides at or around the mouse start site are not conserved in the human sequence or that unidentified factors controlling initiation site selection bind at different positions. The construction of promoter chimeras will be useful to distinguish between these two possibilities.
We have analyzed the proximal P1 and P2 promoter regions and the 5'-untranslated region and signal sequence exons of genomic DNA from PHP1b patients for defects that might lead to disruption of the tissue-specific expression of the PTHR gene. In contrast to PHP1a, in which multiple endocrine systems may malfunction in addition to pathways controlled by PTH, functional hypoparathyroidism is the sole phenotype observed in PHP1b. Therefore, the locus of mutation in this latter disorder appears to be specific to the PTH signaling pathway. In addition, although PTH signaling in the kidney is defective, several reports have documented osteolytic lesions in PHP1b, consistent with the persistence of skeletal responsiveness to high circulating PTH concentrations (34, 35). Consequently, the phenotype associated with PHP1b appears to emanate from a defect in PTH signaling restricted to the kidney. In view of the fact that we have identified multiple promoters controlling PTHR gene expression, the possibility arose that disruption of expression of a PTHR promoter or of the function of a promoter-specific transcript could account for the phenotype of PHP1b.
Southern blotting analysis of the structures of the regulatory regions of the PTHR gene in the seven patients tested has excluded the presence of gross rearrangements or deletions that would disrupt promoter function. Genomic DNA from PHP1b patients amplified by PCR was sequenced to look for more subtle alterations that might affect promoter initiation sites, splice junctions, or transcript stability. No differences were observed in the patterns of restriction sites in these regions or in the sequences of crucial elements. Thus, in none of the seven PHP1b patients tested can the phenotype be accounted for by mutations affecting the initiation, splicing, or stability of transcripts expressed from P1 or P2. Coupled with the lack of mutations in the PTHR-coding region found by other groups, our results suggest that mutations causing PHP1b could reside either elsewhere in the PTHR gene promoter or in the coding or regulatory sequences of another gene. The hPTHR and hPTH2 receptor genes have been mapped to 3p21.1-p22 (36, 37) and 2q33 (38), respectively. Preliminary linkage analyses using polymorphic markers at or near PTHR or PTH2 receptor loci have indicated that mutations in two multiplex families giving rise to PHP1b phenotypes may reside outside of loci encoding the PTHR and PTH2 receptor (38). However, it is important to note that mutations at more than one locus have been shown to account for the phenotype of PHP1a (22, 23, 24). Therefore, we cannot rule out the possibility that disruption of tissue-specific initiation, splicing, or stability of a PTHR transcript may account for PHP1b in some patients.
Candidate loci responsible for PHP1b other than the PTHR may include those encoding transcription factors that would control kidney-specific transcription of the PTHR. There are precedents for deficiencies in such factors. Defects in patients with insulin resistance and noninsulin-dependent diabetes mellitus have been ascribed to markedly reduced levels of transcription factors required for normal expression of the insulin receptor gene (39). To be implicated in PHP1b, such a factor would be required to be not only specific to the kidney, but also to the PTH signaling pathway. Disruption of its expression would not perturb the expression of other genes essential for normal kidney function. Alternatively, the defect may lie in a factor required for PTH-stimulated generation of cAMP, but acting downstream of the receptor or in a factor that antagonizes functional PTH-PTHR interaction. Tissue-specific factors that regulate G protein signaling have been identified (40). However, to be implicated in PHP1b, such factors would have to be specific to PTH signaling and not be important for other pathways. Characterization of factors controlling the expression of PTHR or modulating signaling through the PTHR will, therefore, be essential to fully understand the molecular mechanism of PTH-PTHR signaling and its disruption under pathological conditions.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Royal Victoria Hospital Research Institute
Fellowship.
3 Recipient of a Medical Research Council of Canada studentship.
4 Recipient of a Medical Research Council of Canada Scientist
Award.
5 Chercheur-boursier of the Fonds de Recherche en Santé du
Québec.
Received November 7, 1996.
Revised January 8, 1997.
Accepted January 15, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits. Cell. 86:445–452.[CrossRef][Medline]
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