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Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, National Institutes of Health (S.R.J.H., T.B.U.), Bethesda, Maryland 20892; and Endocrine Unit, Massachusetts General Hospital and Harvard Medical School (D.A.R., H.J.), Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Ted B. Usdin, Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, National Institutes of Health, Building 36, Room 3D06, 36 Convent Drive, MSC4094, Bethesda, Maryland 20892-4094. E-mail: usdin{at}codon.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat and human PTH2 receptors are potently activated by a recently identified peptide, tuberoinfundibular peptide of 39 residues (TIP39) (12). TIP39 has some homology to PTH and PTHrP. Seven of the C-terminal 19 amino acid residues of bovine TIP39 are identical to corresponding residues in bovine PTH, and a number of N-terminal residues are similar (12). PTH is a weak partial agonist at the rat PTH2 receptor, suggesting that it has little role at this receptor (13), but PTH is equipotent with TIP39 at the human PTH2 receptor (12, 13). A PTH2 receptor complementary DNA (cDNA) has recently been isolated from zebrafish (5). Like human and rat PTH2 receptors it is activated by PTH and not by PTHrP. The presence of a PTH2 receptor in zebrafish was initially interpreted as evidence for the existence of a PTH-like molecule before evolution of the parathyroid gland (5). However, cAMP accumulation in response to PTH at the zPTH2 receptor was small compared with the response at zPTH1 and zPTH3 receptors and the human (h) PTH2 receptor (4, 5), and the effect of TIP39 has not been examined. Thus, one aim of this study was to compare the effect of TIP39 on the zPTH2 receptor with those of various PTH peptides.
A third PTH-recognizing receptor (PTH3 receptor) has recently been identified in zebrafish (4). This receptor was initially described as PTHrP selective, based upon the pharmacological profile of amino-terminal fragments of human PTH and mammalian and fugufish PTHrP. The zebrafish PTH3 receptor is of particular interest because of potential physiological functions of a receptor selective for PTHrP. Recent data suggest the presence of a PTHrP-selective receptor in the rat supraoptic nucleus (SON) that stimulates vasopressin release (14, 15). The zPTH3 receptor will obviously be of great utility in identifying this mammalian receptor if it represents a close homolog. The selectivity of the zPTH3 receptor for PTHrP was defined with mammalian (the rat and human sequences are the same) and fugufish PTHrP-(1–36) and hPTH-(1–34) (4). The PTHrP-selective effect in the rat SON was defined using mainly rat PTH-(1–34). Thus, the second aim of this study was to characterize further the ligand selectivity of the zPTH3 receptor and in particular to test the effects of rat PTH peptides on its activation. These data could reflect on the likelihood that a PTH3-like receptor is responsible for the PTHrP-selective effects in the SON.
To address the ligand specificity of the zebrafish PTH receptors we evaluated the effects of the three known types of PTH receptor ligands (PTH, PTHrP, and TIP39) in assays of cAMP accumulation, using COS-7 cells transiently expressing the three receptors. At the zPTH2 receptor, as at the rPTH2 receptor, TIP39 displays high potency and efficacy, whereas the PTH peptides are all partial agonists. These data support the hypothesis that TIP39 is an endogenous ligand for the PTH2 receptor and suggest that there may be a TIP39-like molecule in teleosts. At the zPTH3 receptor, rat PTH-(1–34) is more potent than PTHrP-(1–36) and binds to the receptor with high affinity. With the exception of hPTH, the pharmacological profile of the zPTH3 receptor is thus similar to that of the zPTH1 and mammalian PTH1 receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and transient expression in COS-7 cells
COS-7 cells were grown as previously described (16). For cAMP
accumulation assays, COS-7 cells were transfected as previously
described (16), except for use of 10-cm tissue culture dishes and 10
µg plasmid DNA. The next day the cells were transferred after
trypsinization to 96-well plates at a density of 50,000 cells/well. For
radioligand binding assays, COS-7 cells were plated in 24-well plates
at a density of 250,000 cells/well and transfected with 0.5 µg/well
plasmid DNA. The plasmids used in this study were zPTH1R(FL)/pcDNAI/Amp
(encoding the zPTH1 receptor) (4), zPTH2R/pcDNAI/Amp (encoding the
zPTH2 receptor) (5), zPTH2R(43)/pcDNAI/Amp (encoding a putative splice
variant of the zPTH2 receptor) (5), zeb3-pcDNAI/Amp (encoding the zPTH3
receptor) (4), and pcDNAHAPrP (encoding the human
PTH1 receptor incorporating a C-terminal hemagglutinin tag) (16).
Measurement of cellular levels of cAMP
After removal of medium, transfected COS-7 cells were treated
for 40 min at 37 C with 50 µl/well cAMP assay buffer [DMEM
containing 25 mM HEPES supplemented with 0.1% BSA, 30
µM Ro 20–1724 (Research Biochemicals International, Natick, MA), 100 µM
[4-(2-aminoethyl)]-benzenesulfonylflouride, and 1 µg/ml
bacitracin]. This buffer was removed and replaced with 40 µl fresh
buffer. Test agents were added in a volume of 10 µl, and the
cells were incubated for an additional 40 min at 37 C. The assay was
then terminated by the addition of 50 µl 0.1 N HCl and
0.1 mM CaCl2. cAMP was quantified by
RIA, as previously described (16).
Whole cell radioligand binding assays
Binding of rPTH-(1–34) and bTIP39 was assessed by measuring
inhibition of
[125I][Nle8,21,Tyr34]rPTH-(1–34)
amide binding to COS-7 cells expressing hPTH1, zPTH1, and zPTH3
receptors. Cells in 24-well plates were used 3 days after transfection.
Cells were washed once with binding buffer (50 mM Tris, 100
mM NaCl, 5 mM KCl, and 2 mM
CaCl2, pH 7.5, with HCl, supplemented with 5%
heat-inactivated horse serum, 0.5% FBS, 1 µg/ml bacitracin, and 100
µM [4-(2-aminoethyl)]-benzenesulfonylflouride). To each
well was added sequentially 150 µl binding buffer, 50 µl unlabeled
ligand diluted in binding buffer, and 50 µl radioligand diluted in
binding buffer (
50,000–100,000 cpm/well). Total binding was defined
in the absence of unlabeled ligand, and nonspecific binding was
measured in the presence of 3.2 µM rPTH-(1–34). Cells
were incubated at 15 C for 3 h. The assay plates were then placed
on ice for 10 min and washed twice with 0.5ml/well binding buffer.
Cell-associated radioactivity was extracted with 0.5 ml 1.0
N NaOH. Samples were transferred to tubes, and
radioactivity was measured in a Wallac, Inc. 1470 Wizard
-counter. Nonspecific binding was 3–5% of the total counts added
for all receptors tested. Total binding was 28–47% of the added
radioactivity for the zPTH1 receptor, 17–23% for the zPTH3 receptor,
and 12–33% for the hPTH1 receptor.
Data analysis
Concentration dependence data for ligand-stimulated cAMP
accumulation and inhibition of
[125I][Nle8,21,Tyr34]rPTH-(1–34)
amide binding was analyzed with the following four parameter-logistic
equation using Prism 2.01 (GraphPad Software, Inc., San
Diego, CA): y = min + [(max - min)/(1 + 10
(LogK - X)n)], where
X is the logarithm of the ligand concentration, and
n is Hill slope. For cAMP accumulation data, y is
the amount of cAMP accumulated at a given peptide concentration, min is
the cAMP level in the absence of ligand, max is the maximum level
produced, and LogK is the logEC50. For inhibition
of radioligand binding, y is the counts per min bound at a
given unlabeled ligand concentration, min is nonspecific binding
[measured in the presence of 3.2 µM
rPTH-(1–34)], max is total binding (measured in the absence of
unlabeled ligand), and LogK is the logIC50.
Statistical comparison of multiple means was performed initially by
single-factor ANOVA followed by post-hoc analysis with the
Newman-Keuls test. Statistical comparison of two means was performed
using two-tailed Student’s t test, with statistical
significance specified by P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activity of each of the three known types of PTH receptor ligand
was evaluated by measuring cAMP accumulation in COS-7 cells transiently
expressing the different receptors. At both isoforms of the zPTH2
receptor, TIP39 produced a 2- to 3.5-fold greater maximal stimulation
of adenylyl cyclase than the PTH peptides tested (Fig. 1
and Table 1), an effect that was statistically
significant (P < 0.001, assessed by pairwise
comparison of maximal effect (Emax) values
with the Newman-Keuls test). The rank order of
Emax (intrinsic activity) was similar at both
isoforms of the receptor [TIP39 > rPTH-(1–84) =
rPTH-(1–34) = hPTH-(1–34) = hPTH-(1–84) >
[Tyr36]PTHrP-(1–36) for zPTH2 and TIP39
> rPTH-(1–84) > rPTH-(1–34) = hPTH-(1–34) =
hPTH-(1–84) > [Tyr36]PTHrP-(1–36) for
zPTH2(43); Table 1]. The intrinsic activity values of the peptides
were also similar at both receptor isoforms and similar to those for
the rat PTH2 receptor (Table 1). The maximal amount of cAMP produced by
TIP39 stimulation at the zPTH2 isoform was significantly lower than
that produced at the zPTH2(43) isoform (1.3 ± 0.2 vs.
3.0 ± 0.6 pmol/well, respectively). The maximal TIP39 effect at
the zPTH(43) receptor was not significantly different from that
produced at the rat PTH2 receptor expressed in COS-7 cells (4.3 ±
0.5 pmol/well).
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). The
EC50 value for TIP39 was 5.2 nM at
the zPTH2 receptor and 2.6 nM at the zPTH2(43) isoform
(compared with 0.66 nM at the rat PTH2 receptor). The
potencies of the different PTH peptides varied from 2.6–150
nM at the zPTH2 receptor and from 6.9–40 nM at
the zPTH2(43) isoform. The EC50 values for the
PTH peptides were not well defined in some cases (Table 1) because the
signal to noise ratio in the cAMP RIA was low for these partial
agonists (as little as 1.5-fold in some assays). As a result the rank
order of EC50 for the ligands was poorly defined
by ANOVA and pairwise comparisons of mean EC50
values.
Comparison of PTH and PTHrP activation at zPTH1 and zPTH3
receptors
The zPTH3 receptor is a novel PTH receptor with appreciable
homology to the zPTH1 receptor (61% amino acid sequence identity) (4).
The zPTH3 receptor has previously been shown to be activated with
higher potency by [Tyr36]PTHrP-(1–36) than by
[Tyr34]hPTH-(1–34). In this study we extended
the investigation of ligand activation by including full-length hPTH
[hPTH-(1–84)], rPTH-(1–34), rPTH-(1–84), and TIP39. We also tested
the effect of these ligands on the zPTH1 receptor to obtain a detailed
comparison of zPTH1 and zPTH3 receptor activation.
At the zPTH3 receptor, hPTH-(1–34) displayed lower potency than
[Tyr36]PTHrP-(1–36), as reported previously
(Fig. 2 and Table 2). However, rPTH-(1–34) was 7-fold more
potent than [Tyr36]PTHrP-(1–36) (Fig. 2 and
Table 2). These differences were significant (P <
0.05) as assessed by pairwise comparison of the mean
EC50 values. Using this analysis, the rank order
of potency at the zPTH3 receptor was rPTH-(1–34) =
rPTH-(1–84) > [Tyr36]PTHrP-(1–36)
> hPTH-(1–34) > hPTH-(1–84). The general activation profile of
the zPTH3 receptor was similar to that of the zPTH1 receptor (Fig. 2
and Table 2): 1) All of the PTH peptides and the PTHrP analog were full
agonists for stimulation of adenylyl cyclase at both receptors. 2)
TIP39 produced little or no stimulation at either receptor. 3) The
N-terminal fragments rPTH-(1–34) and hPTH-(1–34) were equivalently
potent or more potent than full-length PTH of the corresponding
species, indicating that the N-terminal fragment of PTH is sufficient
for full and potent activation of adenylyl cyclase at these receptors.
4) The difference between mean ligand potency at the zPTH1 and zPTH3
receptors was less than 10-fold, with the exception of hPTH-(1–34),
which was 18-fold more potent at the zPTH1 receptor (Table 2). The
maximal hPTH-(1–34)-stimulated cAMP level at zPTH1 and zPTH3 receptors
was not significantly different (2.5 ± 0.5 and 3.3 ± 0.6
pmol/well, respectively). Neither of these values was significantly
different from the hPTH-(1–34) Emax at the human
PTH1 receptor expressed in COS-7 cells (3.8 ± 0.4 pmol/well)
(12).
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). The IC50 for
TIP39 at the zPTH3 receptor (350 nM) was 8-fold lower than
that at the zPTH1 receptor and approximately equal to that at the human
PTH1 receptor (Table 3). rPTH-(1–34)
bound with high affinity (3–14 nM) to all three of these
receptors (Fig. 3 and Table 3). The IC50 for
rPTH-(1–34) at the human PTH1 receptor (14 nM) was
comparable with that determined previously (16). The affinity of TIP39
for the human PTH1 receptor in whole COS-7 cells (350 nM)
was similar to that measured for the receptor in isolated membranes
prepared from these cells (160 nM; graphical data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At both isoforms of the zPTH2 receptor, bTIP39 displays high efficacy and potency despite the considerable evolutionary distance between the cow and the zebrafish. This observation strongly suggests that TIP39 is an endogenous ligand for the PTH2 receptor. The existence of the zebrafish PTH2 receptor led initially to the suggestion that PTH is present in bony fish, as this receptor is activated by PTH, but not by PTHrP (5). However, as TIP39 is a better agonist at the receptor, the existence of the zPTH2 receptor can be interpreted to suggest the presence of a TIP39-like molecule in this species rather than lending support to the presence of PTH. The possibility that a teleost homolog of PTH might display a much higher efficacy than mammalian PTH cannot be ruled out. However, mammalian PTH strongly activates zPTH1 and zPTH3 receptors, suggesting that a species difference minimally affects the efficacy of PTH at these receptors. Overall, the profile of ligand activation of the zPTH2 receptor isoforms closely resembles that of the rPTH2 receptor, for which PTH peptides are also partial agonists (13). This similarity suggests that the PTH2 receptor has been functionally well conserved through evolution. However, PTH is a full agonist at the hPTH2 receptor (12, 13). One possible view is that the PTH2 receptor has mediated the effects of TIP39 through evolution and that in humans the PTH2 receptor has gained full responsiveness to PTH.
The zebrafish PTH3 receptor has previously been demonstrated to be selective for mammalian and teleost PTHrP over human [Tyr34]PTH-(1–34). The hPTH ligand was 21-fold less potent for stimulation of cAMP accumulation than [Tyr36]PTHrP-(1–36) and bound to the receptor with a 56-fold lower affinity (4). This profile was of interest because a PTHrP-selective receptor that stimulates vasopressin release has been demonstrated pharmacologically in rat SON. In SON slices, PTHrP-(1–34) stimulated vasopressin release and cAMP accumulation. [125I][Tyr34]PTHrP-(1–34) bound specifically to membranes prepared from the SON, and the binding was inhibited by unlabeled PTHrP-(1–34) (14). Finally, centrally administered PTHrP-(1–34) stimulates vasopressin release in vivo (15). In all of these experiments, PTHrP selectivity was demonstrated by comparison with rPTH-(1–34), which had little or no effect. We therefore examined the effect of rPTH-(1–34) at the zPTH3 receptor. In cAMP accumulation assays, rPTH-(1–34) was significantly more potent than [Tyr36]PTHrP-(1–36) at the zPTH3 receptor. In addition, rPTH-(1–34) displayed high affinity for this receptor in radioligand binding assays (IC50 = 3.2 nM). These data indicate that unless the PTH3 receptor is much less functionally conserved than the PTH1 and PTH2 receptors, this receptor is unlikely to mediate the PTHrP-selective effect observed in the rat SON. We found that the [Tyr36]PTHrP selectivity over hPTH-(1–34) at the zPTH3 receptor was only 3-fold, which is less than that previously reported (21-fold) (4). This difference may have resulted from the use of a slightly different ligands; hPTH-(1–34) and [Tyr36]PTHrP-(1–36) were used in this study, whereas [Tyr34]hPTH-(1–34) amide and [Tyr36]PTHrP-(1–36) amide were used previously. In addition, the previous study employed different cell culture conditions after transfection (to maximize receptor number), which could have contributed to the higher potency observed for [Tyr36]PTHrP-(1–36). Overall, the profile of the zPTH3 receptor for stimulation of adenylyl cyclase resembles those of zebrafish, frog, and mammalian PTH1 receptors (1, 2, 4, 17). However, these receptors may differ significantly in coupling to production of inositol phospholipids; ligand activation of the zPTH1 receptor stimulates inositol phospholipid accumulation in COS-7 cells, whereas the zPTH3 receptor is not detectably coupled to this second messenger pathway.
A teleost PTHrP molecule is a candidate for an endogenous ligand of the zPTH1 and zPTH3 receptors (4). PTHrP-like immunoreactivity and messenger RNA have been detected in several fish species (18, 19, 20, 21), and a PTHrP-like sequence has been identified in the pufferfish genome (FUGU Landmark Mapping Project Database clones 115E01AC6 and 155E01eB5). The presence of PTH in teleosts remains to be conclusively demonstrated. A short PTH-like DNA sequence encoding 10 amino acids has been amplified from rainbow trout genomic DNA (22), and PTH-like immunoreactivity has been described in teleosts (18, 19). Although the pharmacological profiles of the zebrafish receptors do not exclude the existence of teleost PTH, the presence of these receptors does not provide a strong argument for the presence of this peptide, as all three receptors respond equally well or better to other ligands.
The family of PTH receptors and the family of PTH-like peptides has expanded dramatically in the last several years with the discovery of PTH2 and PTH3 receptors and TIP39. The isolation of all three receptors from zebrafish provides the means to study the evolution and biological roles of these receptors. The receptors should also prove useful for studying the structural basis of ligand selectivity. The comprehensive pharmacological evaluation of the receptors in this study provides a sound basis for the further investigation of this diverse, physiologically important receptor and ligand family.
Received February 1, 2000.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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), and
hPTH-(1–84) (
). C and D, Receptor activation by rPTH-(1–34) (
),
rPTH-(1–84) (
), and [Tyr36]PTHrP-(1–36) (
). The
data were fitted to a four-parameter logistic equation using nonlinear
regression to obtain estimates of EC50 and Emax
(Table 1
