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Endocrine Unit Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nt) that lacks most
of the amino-terminal extracellular domain. The binding of PTH(1–14)
to LLC-PK1 or COS-7 cells transfected with the
intact PTH-1 receptor was too weak to detect; however, PTH(1–14)
dose-dependently stimulated cAMP formation in these cells over the dose
range of 1–100 µM. PTH(1–14) also
stimulated cAMP formation in COS-7 cells transiently transfected with
rNt, and its potency with this receptor was nearly equal to that
seen with the intact receptor. In contrast, PTH(1–34) was 
100-fold
weaker in potency with rNt than it was with the intact receptor.
Alanine scanning of PTH(1–14) revealed that for both the intact and
truncated receptors, the 1–9 segment of PTH forms a critical receptor
activation domain. Taken together, these results demonstrate that the
amino-terminal portion of PTH(1–34) interacts with the juxtamembrane
regions of the PTH-1 receptor and that these interactions are
sufficient for initiating signal transduction. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The PTH-1 receptor is a member of the family B subgroup of G protein-coupled receptors and is thus related to the receptors for calcitonin, secretin, glucagon, and several other peptide hormones (16, 17, 18, 19). Site-directed mutagenesis and chimera studies have identified several regions of the PTH-1 receptor that modulate ligand interaction, and these are located in the large amino-terminal extracellular domain and the region containing the extracellular loops and transmembrane helices (20, 21, 22, 23). Studies on PTH receptor/calcitonin receptor chimeras and PTH/calcitonin hybrid ligands have suggested that the carboxyl-terminal (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) binding region of the ligand interacts with the receptor’s amino-terminal extracellular domain, and that the amino-terminal signaling portion of the ligand interacts with the receptor region containing the extracellular loops and membrane-spanning helices (24). Similar conclusions were formed based on studies of calcitonin/glucagon receptor chimeras (25). Recent results from cross-linking studies performed with PTH or PTHrP analogs containing the photoreactive benzophenone moiety at various positions are concordant with the general hypothesis stated above (26, 27, 28). However, questions remain about the mechanism of complex formation and action. For example, hormone binding to the amino-terminal extracellular domain may need to precede the functional interaction with the juxtamembrane region, as has been postulated for the glycoprotein hormone receptors (19). Additionally, it is unclear whether residues in the (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) domain of PTH contribute to cAMP signaling in addition to binding.
As a means to simplify the analysis of the interaction of PTH with the PTH-1 receptor, we are utilizing a domain-based approach that involves the use of small active portions of the hormone and receptor. As described herein, we use this approach to analyze the functional interaction of short amino-terminal PTH ligands and a PTH receptor mutant that lacks most of the amino-terminal extracellular domain. The results demonstrate that the conserved (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) segment of PTH functions as an autonomous signaling peptide, and that the interaction of this peptide with the juxtamembrane region of the receptor is sufficient for second-messenger signaling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 x
106 receptors per cell) of the cloned human PTH-1 receptor
(29). As shown in Fig. 1A
, the control
peptide PTH(1–34) mediated a 50-fold increase in intracellular cAMP
levels relative to the basal cAMP level, and the estimated
EC50 for this response was 2 nM. With
PTH(1–13) and shorter fragments, little or no increase in cAMP
accumulation was observed (Fig. 1A). Two amino-terminal fragments,
PTH(1–15), stimulated cAMP formation to about 20-fold
over the basal level; the doses required for these responses were 5 to
6 orders of magnitude higher than the dose required for an equivalent
response with PTH(1–34). Parental LLC-PK1 cells, which do
not express PTH receptors, were unresponsive to PTH(1–34) or
PTH(1–14) (Fig. 1B).
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). With the exception of positions 3
(serine) and 1 (alanine, and therefore not tested in this experiment)
alanine substitutions at most sites in the 1–9 segment of rat
PTH(1–14) yielded peptides that were barely active or inactive. In
contrast, substitutions in the 10–14 region yielded activities that
were comparable with that of native rat PTH(1–14).
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Nt) in which residues 26–181 of the amino-terminal extracellular
domain were deleted. Since the PTH-1 receptor is predicted to be
cleaved by signal peptidase between Ala22 and
Tyr23 (30), and the junction of the N-terminal domain and
transmembrane helix 1 is predicted to be at or near Ile190
(31), rNt is predicted to contain only a short amino-terminal
extracellular segment consisting of residues 23–26 joined to residues
182–190 (Fig. 3B). In COS-7 cells
expressing the intact rat PTH-1 receptor (rWT-HA), PTH(1–34) and
PTH(1–14) mediated cAMP responses that were similar to their responses
in HKRK-B7 cells; thus, PTH(1–14) stimulated a 15-fold increase in
cAMP formation, and its potency was 4 to 5 orders of magnitude weaker
than that of PTH(1–34) (Fig. 3C). Both peptides also stimulated cAMP
formation with rNt, but whereas PTH(1–14) was equipotent with
rWT-HA and rNt, PTH(1–34) was 2 orders of magnitude weaker with
rNt than it was with rWT-HA (compare panels C and D of Fig. 3).
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Nt and rWT-HA suggests that the truncated receptor is well
expressed on the cell surface. This was confirmed by experiments
performed on a similarly truncated receptor derived from rNt that
had an epitope tag (HA) joined C-terminally via a tetraglycine linker
to the short extracellular segment extending from transmembrane helix
1. This mutant receptor, called rNt-HA, exhibited signaling
responses to PTH(1–14) that were equivalent to those
seen for rNT and was expressed on the surface of COS-7 cells at
55% of the expression level seen for rWT-HA, as judged by
antibody-binding analysis (data not shown).
We then used the alanine-scanning set of PTH(1–14) analogs to examine
whether the ligand residues that are required for function with the
truncated receptor differ from those required for function with the
intact receptor. As shown in Fig. 4, A
and B, the cAMP activity profile obtained with these analogs and rNt
mirrored that obtained with rWT-HA (Fig. 4, A and B).
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Nt retained the same
recognition specificity as the corresponding parent ligand and receptor
by performing cross-reactivity experiments using the cloned rat
secretin receptor and secretin ligands. COS-7 cells transfected with
the secretin receptor exhibited a 50-fold increase in cAMP levels in
response to secretin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) (1 µM) but did not respond to
either PTH(1–14) (100
µM) (Fig. 5C). Cells
expressing rNt responded to PTH(1–14) but not to
secretin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) (1 µM) or secretin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) (1
µM) (Fig. 5B). A slight response could be detected for
secretin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) (100 µM) with the secretin receptor and
with rWT-HA.
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Nt. With rWT-HA,
[Leu11,D-Trp12]hPTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2
reduced the efficacy of both PTH(1–34) by as much as
70%, as compared with the responses elicited by these agonists in the
absence of inhibitor (Fig. 6 A). In
contrast, the PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) analog had little or no effect on the
ability of PTH(1–14) to stimulate cAMP production in
cells expressing rNt.
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Ala and Gln37 Ala, are
at the extreme amino terminus of the receptor (26), and the third,
Arg186 Ala, is near the predicted C-terminal end of the
amino-terminal extracellular domain (27). Each of the three full-length
mutant receptors was fully expressed on the cell surface, as judged by
antibody-binding analysis (Ref. 26 and data not shown). The
Thr33 Ala and Gln37 Ala mutations had
no effect on the activity of PTH(1–14), whereas the Arg186
Ala mutation abolished PTH(1–14) activity (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), as
was PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) (data not shown). These observations suggest that
particular structural elements are required for activity in such
truncated peptide ligands. Another point to consider is that some of
the early studies on PTH fragments used cell lines or membranes
prepared from tissues that expressed relatively low levels of
endogenous PTH receptors (12, 14, 15), and these may not provide the
same sensitivity as transfected cells expressing high levels of
receptors, such as we used here. The possibility that the activity we
observed for PTH(1–34) was
precluded by certain observations that serve as internal controls.
First, the potency of PTH(1–14) was unaffected by the truncation of
the receptor, whereas the potency of PTH(1–34) was markedly reduced
with this receptor mutant. Second, the alanine-scanning profile
obtained with our PTH(1–14) analogs closely resembles the
alanine-scanning profile obtained in an independent study conducted by
another research group in which PTH(1–32).
Third, PTH(1–14) did not activate the PTH-2 receptor subtype (data not
shown). Finally, we found equivalent potencies for different
preparations of the native PTH(1–14) peptide synthesized on separate
occasions.
In our studies with the intact receptor, the signaling potency of
PTH(1–14) was about 5 orders of magnitude weaker than that of
PTH(1–34). This reduced potency is not surprising given the absence of
the important receptor-binding residues located in the (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) domain
of the ligand (6, 7, 8, 9). Consistent with this, the binding of PTH(1–14)
to the receptor was too weak to permit detection in heterologous
competition binding assays performed with 125I-PTH(1–34)
as a tracer radioligand and unlabeled PTH(1–14) as a competitor ligand
at final concentrations as high as 3 mM. We also could not
detect direct binding of a radiolabeled PTH(1–14) analog,
[Nle8,Tyr14]-rPTH(1–14)NH2, that
was equipotent to native rat PTH(1–14) in cAMP assays. The inability
to detect binding of PTH(1–14), even at doses that elicit robust
increases in cAMP, most likely reflects the lower sensitivity of the
binding assay, as compared to the cAMP assay. The former assay requires
the ligand-receptor dissociation rate to be slow enough to be
compatible with the rinsing steps involved in separating bound and free
radioligand (33). The signaling assay is less dependent on the ligand
dissociation rate, because the presence of the phosphodiesterase
inhibitor [3-isobutyl-1-methylxanthine (IBMX)] allows cAMP to
accumulate in the cell throughout the assay period, and this
facilitates signal detection. We note that several other studies on
mutant family B receptors have reported agonist-mediated cAMP responses
in the absence of detectable ligand binding (24, 34). Moreover, in the
present study we show that PTH(1–34) is capable of stimulating rNt,
even though ligand binding is too weak to measure (35).
In contrast to the similar potency that PTH(1–14) exhibited with
rNt and rWT-HA, PTH(1–34) was 100-fold weaker with rNt than it
was with the intact receptor (Fig. 3, C and D). This weaker potency is
likely to reflect the loss of important binding interactions that have
been hypothesized to occur between the (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) domain of PTH(1–34)
and the amino-terminal extracellular domain of the receptor (20, 26).
However, the hypothesis that the 15–34 region of PTH binds to the
amino-terminal extracellular domain of the receptor does not exclude
the possibility that the 15–34 region, which by itself does not
stimulate cAMP formation (data not shown), also interacts with the
receptor region containing the extracellular loops and
transmembrane domains. In fact, the 100-fold greater potency
[relative to PTH(1–34) exhibits with rNt (Fig. 3D) could be explained by such interactions. At present, we cannot
exclude alternative possibilities, e.g. the 15–34 domain of
PTH(1–34) might stabilize a favorable secondary structure in the
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) segment and thereby enhance the intrinsic signaling activity of
the amino-terminal residues.
PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) strongly inhibited stimulation of rWT- HA by PTH(1–14),
even though the antagonist was at a 10-fold molar deficiency compared
with the agonist. This result was somewhat unexpected, given that
PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) antagonism requires residues 26–181 in the receptor’s
amino-terminal extracellular domain (Fig. 6B) and that PTH(1–14)
activity does not. It is possible that PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) antagonizes
PTH(1–14) through a competitive mechanism involving overlap in the
receptor sites used by PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and PTH(1–14), perhaps at the
small portion of the receptor’s N-terminal domain retained in rNt
(e.g. residues 182–190). Arginine-186 is important for
PTH(1–14) activity (Fig. 7) and has been shown to be required for the
covalent cross-linking of a PTH(1–34) analog containing a photolabile
modification at position 13 (27). An alternative explanation for the
PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) antagonism of PTH(1–14) invokes a noncompetitive
inhibition mechanism involving allosteric changes in the activation
state of the receptor. This latter possibility is suggested by the
ability of PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) to function as an inverse agonist with
constitutively active PTH receptors (36). However, at present we cannot
distinguish between these two possibilities.
Ligand-specificity determinants in the family B peptide hormone
receptors have been identified in several receptor domains (21, 34, 37, 38, 39, 40). As it seemed possible that the truncated PTH receptor and PTH
fragments used in our study might exhibit relaxed specificity, we
performed a cross-reactivity experiment using secretin ligands and the
secretin receptor. No evidence for cross-reactivity was observed. It is
also noted that PTH(1–14) did not activate the endogenous calcitonin
receptors in untransfected LLC-PK1 cells (Fig. 1B). These
findings suggest that the correct domain structures are preserved in
the PTH(1–14) fragment and the truncated PTH receptor; however, a more
direct analysis of conformation would be required to confirm this. A
moderate response was observed for secretin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) (100
µM) acting on the secretin receptor and on the intact
PTH-1 receptor. Although we have not studied this activity further, it
may be that short amino-terminal fragments of secretin or other family
B ligands could be useful in dissecting ligand interaction sites in
these related receptors.
Our results show that a major portion of the amino-terminal
extracellular domain of the PTH-1 receptor is not essential for
ligand-dependent signal transduction. For most family B receptors, this
domain appears to play an important role in ligand binding (41, 42).
However, there are now several other reports on family B receptors that
indicate that an intact N-terminal domain is not essential for
expression or transmembrane signaling. Large amino-terminal deletions
in the calcitonin receptor (43) and GH-releasing factor receptor (44)
were compatible with surface expression. Moreover, a glucagon receptor
lacking the amino- terminal extracellular domain and containing an
activating mutation in helix 2 (His178 Arg) exhibited
constitutive cAMP-signaling activity (45). In these studies, however,
evidence that the truncated receptors could interact with ligand was
not reported. In a separate study on the lutropin receptor, a
glycohormone receptor belonging to the family A group of G
protein-coupled receptors, it was observed that a receptor mutant
lacking the large amino-terminal extracellular domain could mediate a
cAMP response to high doses of hCG (46). It appears, therefore, that
for at least some of the peptide hormone receptors, the portion of the
receptor containing the seven-transmembrane helices and connecting
loops can function autonomously, with respect to surface expression,
ligand interaction, and G protein coupling.
That the activity of PTH(1–14) was not affected by the deletion of
most of the amino-terminal domain of the PTH-1 receptor suggests that
this ligand interacts predominantly with the portions of the receptor
that are predicted to be close to the membrane, and not with residues
further toward the N terminus of the receptor. This conclusion is
supported by the alanine-scanning experiments performed on PTH(1–14)
in which the profile of tolerant and intolerant residues observed with
rNt closely resembled that seen with the intact receptor.
Furthermore, mutations near the N terminus of the receptor that impair
the binding of PTH(1–34) (Gln37 Ala
and Thr33 Ala) (26) did not affect the activity of
PTH(1–14), whereas the Arg186 Ala mutation at the
C-terminal end of the extracellular N-terminal domain abolished
PTH(1–14) activity. Interestingly, we could not detect an effect of
the Arg186 Ala mutation on PTH(1–27) also did not detect an effect of this
mutation on PTH(1–34) signaling, although the maximum binding of
radiolabeled PTH(1–34) was reduced to 30% of the binding seen for the
WT receptor. One interpretation of the results is that the
Arg186 Ala mutation causes a loss of binding
interactions to the (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) region of PTH, and thus affects a larger
proportion of the total interactions used by PTH(1–14), as compared to
PTH(1–34). Consequently, the mutation has a greater impact on the
signaling potency of the shorter ligand.
The pattern of critical and noncritical residues observed in our alanine scanning of the PTH(1–14) fragment closely matches the patterns found previously in an alanine scan of PTH(1–32). The substitutions in the longer PTH peptide could potentially alter tertiary interactions that might occur between the N- and C-terminal domains of the ligand, such as those suggested by other studies (47, 48, 49, 50). Our current studies on PTH(1–14) indicate that the mutational intolerance of the residues in the (1, 2, 3, 4, 5, 6, 7, 8, 9) segment of PTH cannot be based solely on long-range tertiary interactions with the C-terminal 15–34 domain of the ligand. Instead, these residues must have some local role in function, e.g. they could be involved in a lock-and-key-type interaction with the receptor. Further work is needed to more precisely identify the function of these N-terminal residues of PTH.
The results presented here provide new clues for understanding how PTH interacts with the PTH-1 receptor. A much smaller region of PTH is shown to be sufficient for receptor activation, and this could significantly reduce the complexity of a systematic structure/activity analysis of the hormone’s bioactive region. Domain-based minimization strategies that have been successful for other proteins (51, 52) could conceivably be applied to functional fragments of PTH as an approach for identifying new low molecular weight PTH receptor agonists. Furthermore, the use of smaller PTH ligands and PTH receptors may help simplify the problem of determining the location and functional importance of interactions that occur between this peptide hormone and its receptor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture and DNA Transfection
COS-7 and HKRK-B7 cells were cultured at 37 C in DMEM
supplemented with FBS (10%), penicillin G (20 U/ml), streptomycin
sulfate (20 µg/ml), and Amphotericin B (0.05
µg/ml) in a humidified atmosphere containing 5% CO2.
Stock solutions of EGTA/trypsin and antibiotics were from Gibco BRL (Gaithersburg, MD); FBS was from HyClone Laboratories, Inc. (Logan, UT). The HKRK-B7 cell line was established
previously (29) by stable transfection of LLC-PK1 cells
with a pCDNA-1-based plasmid (Invitrogen, San Diego, CA)
encoding the hPTH-1 receptor (53), and these cells express
approximately 1 x 106 PTH-binding sites per cell. The
HKRK-B7 cells were subcultured in 24-well plates and used for
functional assays 24–72 h after the cell monolayer became confluent.
Transient transfections of COS-7 cells were performed using
diethylaminoethyl-dextran as described previously (39). COS-7
cells were transfected in 24-well plates when the cells were 85–95%
of confluency using 200 ng of plasmid DNA that was purified by cesium
chloride/ethidium bromide gradient centrifugation for each well. Assays
were conducted 72–96 h after transfection. Under these conditions
about 20% of the COS-7 cells become transfected and express about
5 x 106 surface PTH receptors per cell at the time of
assay (39). Both COS-7 and HKRK-B7 cells were shifted to a humidified
incubator containing 5% CO2 set at 33 C 16–24 h before
assay.
Receptor Mutagenesis and Expression
The construction and initial characterization of the
pCDNA-1-based plasmids encoding either the intact epitope-tagged rat
PTH-1 receptor (rWT-HA) or the truncated rPTH-1 receptor have been
described previously (35). The HA tag in rWT-HA is a nine-amino
sequence that replaces residues 93–101 in the receptor’s
extracellular domain and which does not affect receptor function (35).
The truncated receptor, referred to herein as rNt, was originally
referred to as rE1-G (35). This receptor is deleted for exon E1
through exon G (residues 26–181) and, assuming that signal peptidase
cleavage occurs between Ala22 and Tyr23 (30),
is predicted to have for its N terminus residues
Tyr23-Ala24-Leu25 joined to
Glu182 (Fig. 3B). A similar truncated receptor with an
amino-terminal epitope tag, rNt-HA, was constructed by
oligonucleotide-directed mutagenesis (54) using a 96-base mutagenic
primer and single-stranded uracil-containing template DNA derived from
rNt. The resulting mutant receptor has the nine-amino acid HA tag
joined to a tetraglycine linker (Y-P-Y-D-V-P-D-Y-A-G-G-G-G-) inserted
between Ala22 and Glu182 of the rat PTH-1
receptor. Antibody binding to intact COS-7 cells in 24-well plates was
assessed using the monoclonal antibody 12CA5 (Boehringer Mannheim, Indianapolis, IN) and an 125I-labeled
secondary antibody (New England Nuclear, Boston, MA), as described
previously (22).
The mutant rat PTH-1 receptors containing the Thr33 Ala
and Gln37 Ala mutations were derived from rWT-HA and
have been described previously (26). The Arg186 Ala
mutation, recently reported by Adams et al. (27), was
introduced here into rWT-HA by oligonucleotide-directed mutagenesis
(54). The DNA sequences of candidate mutant plasmids were verified in
an 800-nucleotide region spanning the mutation site using the
PE Applied Biosystems Taq DyeDeoxy Terminator
cycle sequencing method, with sample analysis being performed on an ABI
377 PRISM automated sequencer (PE Applied Biosystems,
Foster City, CA). For each mutant receptor, at least two independently
derived plasmids with correct sequences were functionally analyzed and
demonstrated to have identical phenotypes.
Intracellular cAMP
Transfected COS-7 or HKRK-B7 cells were rinsed with 500 µl of
binding buffer (50 mM Tris-HCl, pH 7.7, 100 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 5%
heat-inactivated horse serum, 0.5% heat-inactivated FBS) and 200 µl
of IBMX buffer (DMEM containing 2 mM IBMX, 1 mg/ml BSA, 35
mM HEPES-NaOH, pH 7.4), and 100 µl of binding buffer or
binding buffer containing various amounts of peptide were added. The
plates were incubated for 60 min at room temperature. The buffer was
then withdrawn and the cells were frozen on dry ice, treated with 0.5
ml of 50 mM HCl, and refrozen. After thawing, the lysate
was diluted 30-fold in dH2O, and an aliquot was analyzed
for cAMP content by RIA using unlabeled cAMP as a standard.
For cAMP inhibition assays, transfected COS-7 cells were rinsed once with 500 µl of binding buffer, and 200 µl of IBMX buffer and 100 µl of binding buffer or binding buffer containing the antagonist [Leu11,D-Trp12]hPTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 (10 µM) were added. After a 5-min incubation at room temperature, 10 µl of binding buffer containing PTH(1–14) or PTH(1–34) (agonist peptide) were added, and the incubation was continued for an additional 30 min. The cells were then lysed and intracellular cAMP levels were measured as described above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was funded by NIH Grant DK-11794.
Received for publication December 28, 1998. Revision received February 8, 1999. Accepted for publication February 15, 1999.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M. Castro, V. O. Nikolaev, D. Palm, M. J. Lohse, and J.-P. Vilardaga Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism PNAS, November 1, 2005; 102(44): 16084 - 16089. [Abstract] [Full Text] [PDF]
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J.-R. Barbier, T. J. Gardella, T. Dean, S. MacLean, Z. Potetinova, J. F. Whitfield, and G. E. Willick Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone: EVIDENCE FOR BINDING TO BOTH THE N-TERMINAL EXTRACELLULAR DOMAIN AND EXTRACELLULAR LOOP REGION J. Biol. Chem., June 24, 2005; 280(25): 23771 - 23777. [Abstract] [Full Text] [PDF]
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 T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
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N. Shimizu, T. Dean, J. C. Tsang, A. Khatri, J. T Potts Jr, and T. J. Gardella Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor J. Biol. Chem., January 21, 2005; 280(3): 1797 - 1807. [Abstract] [Full Text] [PDF]
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 R. C. Gensure, B. Ponugoti, Y. Gunes, M. R. Papasani, B. Lanske, M. Bastepe, D. A. Rubin, and H. Juppner Identification and Characterization of Two Parathyroid Hormone-Like Molecules in Zebrafish Endocrinology, April 1, 2004; 145(4): 1634 - 1639. [Abstract] [Full Text] [PDF]
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V. Wittamer, F. Gregoire, P. Robberecht, G. Vassart, D. Communi, and M. Parmentier The C-terminal Nonapeptide of Mature Chemerin Activates the Chemerin Receptor with Low Nanomolar Potency J. Biol. Chem., March 12, 2004; 279(11): 9956 - 9962. [Abstract] [Full Text] [PDF]
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R. C. Gensure, N. Shimizu, J. Tsang, and T. J. Gardella Identification of a Contact Site for Residue 19 of Parathyroid Hormone (PTH) and PTH-Related Protein Analogs in Transmembrane Domain Two of the Type 1 PTH Receptor Mol. Endocrinol., December 1, 2003; 17(12): 2647 - 2658. [Abstract] [Full Text] [PDF]
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M. Shimada, X. Chen, T. Cvrk, H. Hilfiker, M. Parfenova, and G. V. Segre Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide J. Biol. Chem., August 23, 2002; 277(35): 31774 - 31780. [Abstract] [Full Text] [PDF]
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M. Shimizu, P. H. Carter, A. Khatri, J. T. Potts Jr., and T. J. Gardella Enhanced Activity in Parathyroid Hormone-(1-14) and -(1-11): Novel Peptides for Probing Ligand-Receptor Interactions Endocrinology, July 1, 2001; 142(7): 3068 - 3074. [Abstract] [Full Text] [PDF]
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P. H. Carter, M. Shimizu, M. D. Luck, and T. J. Gardella The Hydrophobic Residues Phenylalanine 184 and Leucine 187 in the Type-1 Parathyroid Hormone (PTH) Receptor Functionally Interact with the Amino-terminal Portion of PTH-(1-34) J. Biol. Chem., November 5, 1999; 274(45): 31955 - 31960. [Abstract] [Full Text] [PDF]
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P. H. Carter, H. Jüppner, and T. J. Gardella Studies of the N-Terminal Region of a Parathyroid Hormone-Related Peptide(1-36) Analog: Receptor Subtype-Selective Agonists, Antagonists, and Photochemical Cross-Linking Agents Endocrinology, November 1, 1999; 140(11): 4972 - 4981. [Abstract] [Full Text]
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M. Shimizu, P. H. Carter, and T. J. Gardella Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand J. Biol. Chem., June 23, 2000; 275(26): 19456 - 19460. [Abstract] [Full Text] [PDF]
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S. R. J. Hoare, T. J. Gardella, and T. B. Usdin Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor. EFFECT OF RECEPTOR-G-PROTEIN INTERACTION ON THE LIGAND BINDING MECHANISM AND RECEPTOR CONFORMATION J. Biol. Chem., March 9, 2001; 276(11): 7741 - 7753. [Abstract] [Full Text] [PDF]
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N. Shimizu, J. Guo, and T. J. Gardella Parathyroid Hormone (PTH)-(1-14) and -(1-11) Analogs Conformationally Constrained by alpha -Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor J. Biol. Chem., December 21, 2001; 276(52): 49003 - 49012. [Abstract] [Full Text] [PDF]
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M. Shimizu, J. T. Potts Jr., and T. J. Gardella Minimization of Parathyroid Hormone. NOVEL AMINO-TERMINAL PARATHYROID HORMONE FRAGMENTS WITH ENHANCED POTENCY IN ACTIVATING THE TYPE-1 PARATHYROID HORMONE RECEPTOR J. Biol. Chem., July 14, 2000; 275(29): 21836 - 21843. [Abstract] [Full Text] [PDF]
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