The (114) Fragment of Parathyroid Hormone (PTH) Activates Intact and Amino-Terminally Truncated PTH-1 Receptors
Michael D. Luck,
Percy H. Carter and
Thomas J. Gardella
Endocrine Unit Massachusetts General Hospital and Harvard
Medical School Boston, Massachusetts 02114
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ABSTRACT
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Recent mutagenesis and cross-linking studies
suggest that residues in the carboxyl-terminal portion of PTH(134)
interact with the amino-terminal extracellular domain of the receptor
and thereby contribute strongly to binding energy; and that residues in
the amino-terminal portion of the ligand interact with the receptor
region containing the transmembrane helices and extracellular loops and
thereby induce second messenger signaling. We investigated the latter
component of this hypothesis using the short amino-terminal fragment
PTH(114) and a truncated rat PTH-1 receptor (r
Nt) that lacks most
of the amino-terminal extracellular domain. The binding of PTH(114)
to LLC-PK1 or COS-7 cells transfected with the
intact PTH-1 receptor was too weak to detect; however, PTH(114)
dose-dependently stimulated cAMP formation in these cells over the dose
range of 1100 µM. PTH(114) also
stimulated cAMP formation in COS-7 cells transiently transfected with
r
Nt, and its potency with this receptor was nearly equal to that
seen with the intact receptor. In contrast, PTH(134) was
100-fold
weaker in potency with r
Nt than it was with the intact receptor.
Alanine scanning of PTH(114) revealed that for both the intact and
truncated receptors, the 19 segment of PTH forms a critical receptor
activation domain. Taken together, these results demonstrate that the
amino-terminal portion of PTH(134) interacts with the juxtamembrane
regions of the PTH-1 receptor and that these interactions are
sufficient for initiating signal transduction.
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INTRODUCTION
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PTH, the principal regulator of blood calcium levels, and
PTH-related peptide (PTHrP), a factor that plays a key role in
embryonic bone development and is the causative agent of hypercalcemia
of malignancy, stimulate the type-1 PTH receptor (1, 2, 3). PTH and PTHrP
have potent anabolic effects on bone (4, 5), and so PTH-1 receptor
agonists could be useful in treating metabolic diseases of the
skeleton, such as osteoporosis. In the fully bioactive peptide
PTH(134), the major determinants of receptor-binding affinity reside
within residues 1534 (6, 7, 8, 9), and those of receptor activation lie
within the highly conserved amino-terminal portion. Residues 16 play
a particularly vital role in activation of the adenylyl cyclase
response, and deletion of these residues yields potent competitive
PTH-1 receptor antagonists (10, 11). Amino-terminal PTH or PTHrP
peptide fragments shorter in length than PTH(127) have previously
been found to be biologically inactive (12, 13, 14, 15), although the clear
functional importance and evolutionary conservation of the
amino-terminal residues predicts that they directly interact with
the receptor.
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 receptors 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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PTH(114) Action in Transfected LLC-PK1
Cells
We first determined the minimum length PTH fragment that
stimulated detectable increases in intracellular cAMP levels.
Amino-terminal peptide fragments based on the rat PTH sequence and
ranging in length from PTH(115) were synthesized and
tested for activity in an LLC-PK1-derived cell line called
HKRK-B7 that persistently expresses high levels (
1 x
106 receptors per cell) of the cloned human PTH-1 receptor
(29). As shown in Fig. 1A
, the control
peptide PTH(134) 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(113) and shorter fragments, little or no increase in cAMP
accumulation was observed (Fig. 1A
). Two amino-terminal fragments,
PTH(115), 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(134). Parental LLC-PK1 cells, which do
not express PTH receptors, were unresponsive to PTH(134) or
PTH(114) (Fig. 1B
).

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Figure 1. cAMP-Stimulating Activity of PTH Fragments in
LLC-PK1 Cells
A, Rat PTH(134) analog or amino-terminal rPTH fragments were tested
for cAMP-stimulating activity in an LLC-PK1-derived cell
line (HKRK-B7) stably transfected with the human PTH-1 receptor. Cells
were treated with the peptides indicated in the symbol legend at
various doses for 60 min at 22 C. Intracellular cAMP was measured by
RIA, as described in Materials and Methods. Shown are
combined data (mean ± SEM) from three separate
experiments, each performed in duplicate. B, HKRK-B7 cells or
untransfected LLC-PK1 were treated with rPTH(134) or
rPTH(114), and intracellular cAMP was measured. Shown are data
(mean ± SEM) from a single representative experiment
performed in duplicate.
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To identify residues in the PTH(114) fragment that play a role in
activating the adenylyl cyclase-signaling pathway, we employed an
alanine-scanning approach. Thirteen different alanine-substituted rat
PTH(114) analogs were synthesized and tested for their ability to
stimulate cAMP formation in HKRK-B7 cells (Fig. 2
). With the exception of positions 3
(serine) and 1 (alanine, and therefore not tested in this experiment)
alanine substitutions at most sites in the 19 segment of rat
PTH(114) yielded peptides that were barely active or inactive. In
contrast, substitutions in the 1014 region yielded activities that
were comparable with that of native rat PTH(114).

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Figure 2. Alanine Scan of PTH(114)
HKRK-B7 cells were treated with 100 µM of one of 14
different rPTH(114) analogs, each having a different alanine
substitution at the indicated amino acid position. The resulting cAMP
levels were determined as described in Materials and
Methods. Shown are the combined data (mean ±
SEM) from three separate experiments, each performed in
duplicate. The mean (± SEM) basal cAMP levels observed in
the three experiments was 2.1 ± 0.1 pmol/well, and the maximum
response to rPTH(134) at 0.1 µM was 254 ± 16
pmol/well.
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PTH(114) Action in COS-7 Cells
To investigate the portions of the receptor required for
PTH(114)-mediated signaling, we used a truncated rat PTH-1 receptor
(r
Nt) in which residues 26181 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), r
Nt is predicted to contain only a short amino-terminal
extracellular segment consisting of residues 2326 joined to residues
182190 (Fig. 3B
). In COS-7 cells
expressing the intact rat PTH-1 receptor (rWT-HA), PTH(134) and
PTH(114) mediated cAMP responses that were similar to their responses
in HKRK-B7 cells; thus, PTH(114) stimulated a 15-fold increase in
cAMP formation, and its potency was 4 to 5 orders of magnitude weaker
than that of PTH(134) (Fig. 3C
). Both peptides also stimulated cAMP
formation with r
Nt, but whereas PTH(114) was equipotent with
rWT-HA and r
Nt, PTH(134) was 2 orders of magnitude weaker with
r
Nt than it was with rWT-HA (compare panels C and D of Fig. 3
).
The finding that the potency of PTH(114) was nearly equivalent with
r
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 r
Nt 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 r
Nt-HA, exhibited signaling
responses to PTH(114) that were equivalent to those
seen for r
NT 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(114) 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 r
Nt
mirrored that obtained with rWT-HA (Fig. 4
, A and B).

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Figure 4. Alanine Scan of PTH(114) with Intact and
Truncated PTH Receptors
COS-7 cells transiently transfected with rWT-HA (A) or r Nt (B) were
treated with 100 µM of native rat PTH(114) or 100
µM of a rPTH(114) analog containing a single alanine
substitution for 1 h at 21 C, and the resulting intracellular cAMP
levels were measured by RIA. The amino acid substitutions are indicated
on the axis labels. Peptides were tested in duplicate, and a single
experiment representative of three others is shown.
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Specificity of Truncated Ligands and PTH Receptors
We examined whether PTH(114) and r
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(114) (100
µM) (Fig. 5C
). Cells
expressing r
Nt responded to PTH(114) 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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Figure 5. Specificity of the Truncated Ligand and PTH
Receptor
COS-7 cells transiently transfected with either rWT-HA (A), r Nt (B),
or the intact rat secretin receptor (C) were treated with the indicated
peptides for 60 min at 22 C, and the resulting intracellular cAMP
levels were quantified by RIA. The concentration of peptides present
during the incubations were: rPTH(134), 0.1 µM;
rPTH(127), 1 µM;
and secretin(1 2 3 4 5 6 7 8 9 10 11 12 13 ), 100 µM. Shown are data (mean ±
SEM) from one experiment performed in duplicate, and this
was repeated twice more with equivalent results.
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Inhibition of PTH(134)
As a means to further assess receptor sites of ligand interaction,
we determined whether the amino-terminally truncated antagonist peptide
[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
(11) could block the action of PTH(114) on COS-7 cells expressing
either rWT-HA or r
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(134) 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(114) to stimulate cAMP production in
cells expressing r
Nt.

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Figure 6. Antagonist Properties 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 ) with
PTH(114)
COS-7 cells transfected with rWT-HA (A) or r Nt (B) were treated with
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
(or buffer alone), for 5 min at 22 C, followed by 10 µl of either
rPTH(114) agonist peptide. Incubations were continued
for 30 min at 21 C, and the resulting cAMP levels were measured by RIA,
as described in Materials and Methods. The final
concentration of antagonist peptide present during the incubation was
10 µM; the final concentrations (log) of PTH(134) and
PTH(114) are indicated on the axis label. Shown are data from a
single experiment performed in triplicate. A repeat of the same
experiment yielded equivalent results.
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PTH(114) as a Probe of Mutant Receptors
We tested the possibility that PTH(114) could be used as a
functional probe for analyzing receptor mutations that alter
interaction with PTH(134) ligands. To do this, we used three mutant
rPTH-1 receptors that have substitutions that were previously shown
(26, 27) to modestly impair binding of radiolabeled PTH(134). Two
mutations, Thr33
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(114), whereas the Arg186
Ala mutation abolished PTH(114) activity (Fig. 7
).
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DISCUSSION
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In this study we show that the PTH(114) fragment can stimulate
cAMP formation in cells transfected with the intact PTH-1 receptor or a
truncated PTH-1 receptor lacking most of the amino-terminal
extracellular domain. Although high doses of the peptide were required,
the findings establish that a much smaller region of PTH(134) than
heretofore appreciated can induce receptor activation. It is notable
that we could detect bioactivity with PTH(114) whereas previous
studies on similar PTH or PTHrP fragments, such as PTH(112),
PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) (14), and PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) (13), reported no activity. We also
found that peptides shorter than PTH(114) were inactive (Fig. 1
), 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(134) was
precluded by certain observations that serve as internal controls.
First, the potency of PTH(114) was unaffected by the truncation of
the receptor, whereas the potency of PTH(134) was markedly reduced
with this receptor mutant. Second, the alanine-scanning profile
obtained with our PTH(114) analogs closely resembles the
alanine-scanning profile obtained in an independent study conducted by
another research group in which PTH(132).
Third, PTH(114) did not activate the PTH-2 receptor subtype (data not
shown). Finally, we found equivalent potencies for different
preparations of the native PTH(114) peptide synthesized on separate
occasions.
In our studies with the intact receptor, the signaling potency of
PTH(114) was about 5 orders of magnitude weaker than that of
PTH(134). 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(114)
to the receptor was too weak to permit detection in heterologous
competition binding assays performed with 125I-PTH(134)
as a tracer radioligand and unlabeled PTH(114) as a competitor ligand
at final concentrations as high as 3 mM. We also could not
detect direct binding of a radiolabeled PTH(114) analog,
[Nle8,Tyr14]-rPTH(114)NH2, that
was equipotent to native rat PTH(114) in cAMP assays. The inability
to detect binding of PTH(114), 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(134) is capable of stimulating r
Nt,
even though ligand binding is too weak to measure (35).
In contrast to the similar potency that PTH(114) exhibited with
r
Nt and rWT-HA, PTH(134) was 100-fold weaker with r
Nt 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(134)
and the amino-terminal extracellular domain of the receptor (20, 26).
However, the hypothesis that the 1534 region of PTH binds to the
amino-terminal extracellular domain of the receptor does not exclude
the possibility that the 1534 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(134) exhibits with r
Nt (Fig. 3D
) could be explained by such interactions. At present, we cannot
exclude alternative possibilities, e.g. the 1534 domain of
PTH(134) 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(114),
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 26181 in the receptors
amino-terminal extracellular domain (Fig. 6B
) and that PTH(114)
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(114) 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(114), perhaps at the
small portion of the receptors N-terminal domain retained in r
Nt
(e.g. residues 182190). Arginine-186 is important for
PTH(114) activity (Fig. 7
) and has been shown to be required for the
covalent cross-linking of a PTH(134) 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(114) 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(114) 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(114) 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(114) 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(114)
in which the profile of tolerant and intolerant residues observed with
r
Nt closely resembled that seen with the intact receptor.
Furthermore, mutations near the N terminus of the receptor that impair
the binding of PTH(134) (Gln37
Ala
and Thr33
Ala) (26) did not affect the activity of
PTH(114), whereas the Arg186
Ala mutation at the
C-terminal end of the extracellular N-terminal domain abolished
PTH(114) activity. Interestingly, we could not detect an effect of
the Arg186
Ala mutation on PTH(127) also did not detect an effect of this
mutation on PTH(134) signaling, although the maximum binding of
radiolabeled PTH(134) 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(114), as compared to
PTH(134). 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(114) fragment closely matches the
patterns found previously in an alanine scan of PTH(132). 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(114) 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 1534 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 hormones 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.
 |
MATERIALS AND METHODS
|
---|
Peptides
Peptides were prepared by the Biopolymer Synthesis Facility at
Massachusetts General Hospital (Boston, MA) using solid-phase chemistry
with Fmoc (N-(9-fluorenyl)methoxycarbonyl) protecting groups
and trifluoroacetic acid-mediated deprotection. All peptides were
C-terminally amidated. The PTH(114) analogs were synthesized on a
multiple peptide synthesizer (model 396 MBS, Advanced ChemTech, Inc., Louisville, KY) at 0.025 mM scale. The
completed peptides were desalted by adsorption on a C18 cartridge
(Sep-Pak) and then analyzed by reversed-phase C18-based HPLC,
MALDI-mass spectrometry, and amino acid analysis. The PTH(134)
control peptide,
[Nle8,21,Tyr34]rPTH-(134)NH2,
and 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) antagonist peptide,
[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,
were prepared on an model 431A synthesizer (PE Applied Biosystems, Norwalk, CT) at 0.1 mM scale,
purified by reversed-phase C18-based HPLC and characterized as
described above. Concentrated stock solutions of peptides, 10
mM for PTH(114) analogs and 0.3 mM for
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(134), were prepared in 10 mM acetic
acid, quantified by acid hydrolysis and amino acid analysis, and stored
at -80 C.
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 2472 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 8595%
of confluency using 200 ng of plasmid DNA that was purified by cesium
chloride/ethidium bromide gradient centrifugation for each well. Assays
were conducted 7296 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 1624 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 93101 in the receptors
extracellular domain and which does not affect receptor function (35).
The truncated receptor, referred to herein as r
Nt, was originally
referred to as r
E1-G (35). This receptor is deleted for exon E1
through exon G (residues 26181) 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, r
Nt-HA, was constructed by
oligonucleotide-directed mutagenesis (54) using a 96-base mutagenic
primer and single-stranded uracil-containing template DNA derived from
r
Nt. 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(114) or
PTH(134) (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.
 |
ACKNOWLEDGMENTS
|
---|
We thank John T. Potts Jr., Harald Jüppner, and Henry M.
Kronenberg of the Endocrine Unit at Massachusetts General Hospital
(M.G.H.) for helpful discussions and review of the manuscript and Ashok
Khatri of the M.G.H. Bioploymer Core Facility for the preparation of
peptides.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: Gardella{at}helix.MGH.Harvard.edu
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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