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Endocrine Unit (M.S., P.H.C., A.K., J.T.P., T.J.G.) and Biopolymer Core Facility (A.K.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
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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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Recently, we documented that bioactivity can be detected in short amino-terminal PTH peptide fragments such as PTH-(1–14)NH2 when cells transfected with high numbers of PTH-1 receptors are used for analysis (10). More recently, we extended these studies and reported that certain amino acid substitutions in PTH-(1–14) can enhance activity in an additive way, such that [Ala3,10,12,Arg11,Trp14]PTH-(1–14)NH2 is 200-fold more potent in stimulating cAMP than is native PTH-(1–14)NH2 in the LLC-PK1-derived cell line HKRK-B7 (EC50 = 0.57 ± 0.11 and 133 ± 16 µM, respectively) (11). In this latter study we noted that effects of various substitutions at position 11 in PTH-(1–14)NH2 on activity spanned a wide range; the most positive effects occurred with arginine and lysine, which resulted in peptides that were approximately 150% as active as native PTH-(1–14)NH2, and the most deleterious effects occurred with glutamic acid, histidine, serine, and proline. In the present study we analyzed further the structural basis for the potency-enhancing effects of arginine and lysine substitutions at position 11 in amino-terminal PTH fragment analogs by using nonnatural amino acids that share some structural similarity to arginine or lysine. The study resulted in new PTH-(1–14) and PTH-(1–11) analogs that are considerably more potent than the previously described short amino-terminal fragment peptides. We also show that the activity-enhanced 11-residue peptide can be used as a functional probe to explore the ligand/receptor interface.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The LLC-PK1-derived cell lines HKRK-B7 and
HKRK-B28 are stably transfected with the human P1Rc and express
approximately 950,000 and 280,000 receptors/cell, respectively
(12). These cells as well as SaOS-2 and COS-7 cells were
cultured at 37 C in T-75 flasks (75 mm2) in DMEM
supplemented with FBS (10%), penicillin G (20 U/ml), streptomycin
sulfate (20 µg/ml), and amphotericin (0.05 µg/ml) in a
humidified atmosphere containing 5% CO2. Stock
solutions of EGTA/trypsin and antibiotics were obtained from Life Technologies, Inc.; FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). Cells were subcultured in
24-well plates and, when confluent, were treated with fresh medium and
shifted to 33 C for 12–24 h before assay. COS-7 cells were transiently
transfected with pcDNA-1-based plasmid encoding the intact and mutant
hPTH-1 receptors using diethylaminoethyl-dextran and 200 ng cesium
chloride-purified plasmid DNA/well of a 24-well plate as described
previously (13).
cAMP stimulation
Stimulation of cells with peptide analogs was performed in
24-well plates. Cells were rinsed with 0.5 ml binding buffer (50
mM Tris-HCl, 100 mM NaCl, 5 mM KCl,
2 mM CaCl2, 5% heat-inactivated
horse serum, and 0.5% FBS, adjusted to pH 7.7 with HCl) and treated
with 200 µl cAMP assay buffer (DMEM containing 2 mM
3-isobutyl-1-methylxanthine, 1 mg/ml BSA, and 35 mM
HEPES-NaOH, pH 7.4) and 100 µl binding buffer containing varying
amounts of peptide analog (final volume, 300 µl). The medium was
removed after incubation for 1 h at room temperature, and the
cells were frozen on dry ice, lysed with 0.5 ml 50 mM HCl,
and refrozen (-80 C). The cAMP content of the diluted lysate was
determined by RIA. The EC50 and corresponding
maximum response values (Emax) were calculated
using nonlinear regression (see below).
Stimulation of inositol phosphate production
COS-7 cells were used for these assays because they provided a
greater phospholipase C response to PTH ligands than did the other cell
lines available, including the transfected
LLC-PK1 cells (14). COS-7 cells
transfected as described above with hP1Rc-WT were treated with
serum-free, inositol-free DMEM containing 0.1% BSA and
[3H]myo-inositol (NEN Life Science Products, Boston, MA; 2 µCi/ml) for 16 h before assay. At
the time of the assay, the cells were rinsed with binding buffer
containing LiCl (30 mM) and treated with the same buffer
with or without a PTH analog. The cells were then incubated at 37 C for
40 min, after which the buffer was removed and replaced by 0.5 ml
ice-cold 5% trichloroacetic acid solution. After 3 h on ice, the
lysate was collected and extracted twice with ethyl ether. The lysate
was then applied to an ion exchange column (0.5 ml resin bed), and the
total inositol phosphates were eluted as described previously
(15) and counted in liquid scintillation cocktail.
Competition binding
Binding reactions were performed in 24-well plates.
HKRK-B28 cells were rinsed with 0.5 ml binding buffer, and then treated
successively with 100 µl binding buffer, 100 µl binding buffer
containing various amounts of unlabeled competitor ligand, and 100 µl
binding buffer containing approximately 100,000 cpm
[125I]PTH-(3–34) (
26 fmol; final volume,
300 µl). Incubations were performed for 4 h at 15 C. Cells
were then placed on ice, the binding medium was removed, and the
monolayer was rinsed three times with 0.5 ml cold binding buffer. The
cells were subsequently lysed with 0.5 ml 5 N NaOH and
counted for radioactivity. The nonspecific binding for each experiment
was determined by competition with a 1-µM dose of
unlabeled
[Nle8,21,Tyr34]rPTH-(1–34)NH2.
The maximum specific binding (B0) was the total
radioactivity bound in the absence of unlabeled ligand, corrected for
nonspecific binding. Nonlinear regression was used to calculate binding
IC50 values (see below).
Data calculation
Calculations were performed using Excel (Microsoft Corp., Redmond, WA). Nonlinear regression analyses of binding
and cAMP dose-response data were performed using the four-parameter
equation: yP = Min + [(Max - Min)/(1 +
(IC50/x)slope)].
The Excel Solver function was used for parameter optimization, as
described previously (16, 17). Differences between paired
datasets were statistically evaluated using one-tailed Student’s
t test, assuming unequal variances for the two sets.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1, the rank order of potency for cAMP
stimulation was as follows: homoarginine-11 > arginine-11 >
homophenylalanine-11 > norleucine-11 > citruline-11
leucine-11 (native) > ornithine-11. The
[homoarginine(Har)11]PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) analog was
30-fold more potent than native PTH-(1–14) (P = 0.002)
and 2.6-fold more potent than [Arg11]PTH
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) (P = 0.005). The maximum cAMP responses
attained by peptides containing homoarginine-11, arginine-11,
homophenylalanine-11, and norleucine-11 were comparable to the maximum
cAMP response attained by PTH-(1–34).
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and
Table 2, [Ala3,
12,Gln10,Har11,Trp14]PTH
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) was 1500-fold more potent than native PTH-(1–14) as a cAMP
agonist in HKRK-B28 cells (P = 0.004) and thus only
63-fold weaker than PTH-(1–34) (P = 0.06). A similar
gain in potency was observed for this PTH-(1–14) analog in HKRK-B7
cells (data not shown). The analog [Ala3,
12,Gln10,Har11]PTH-(1–14)
was approximately 3-fold more potent than [Ala3,
12,Gln10,Arg11]PTH-
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) (EC50 = 0.25 ± 0.1 and 0.8 ±
0.3 mM, respectively; P =
0.1); although this effect did not attain statistical
significance, the results suggest that, as in the native peptide
backbone described above, the homoarginine substitution improves
potency by severalfold, relative to arginine, in the modified
PTH-(1–14) backbone. Comparing
[Ala3, 12,Gln10,Har11,Trp14]PTH-
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) and [Ala3, 10,
12,Arg11,Trp14]PTH-(1–14)
revealed that the combined modification of glutamine-10/homoarginine-11
yielded a 12-fold improvement in potency relative to the
alanine-10/arginine-11 combination (P = 0.05).
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and
Table 2
). The homoarginine-11 modification in these PTH-(1–11)
peptides resulted in an approximately 1.5-fold improvement in agonist
potency over the arginine-11 substitution, and the glutamine-10
modification resulted in a 6- to 8-fold improvement in potency relative
to the alanine-10 modification (P = 0.05). The most
potent of these PTH-(1–11) analogs was
[Ala3,Gln10,Har11]PTH-(1–11),
which was 60-fold more potent than unmodified PTH-(1–14)
(P = 0.05).
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20,000 receptors/cell)
(18). In these cells both the modified PTH-(1–14) and
PTH-(1–11) analogs fully stimulated cAMP formation (Fig. 4 and Table 2). The maximum cAMP levels
attained by
[Ala3, 12,Gln10,Har11,Trp14]PTH-(1–14)
and
[Ala3,Gln10,Har11]PTH-(1–11)
exceeded that attained by PTH-(1–34) [141% and 152% of PTH-(1–34),
respectively], but the differences between these maximum responses
were statistically marginal (P = 0.06 and 0.04,
respectively). The signaling potency of
[Ala3, 12,Gln10,Har11,Trp14]PTH-(1–14)
in SaOS-2 cells was 31-fold weaker than it was in HKRK-B28 cells
(3.7 ± 0.4 vs. 0.12 ± 0.04
µM, respectively), whereas the potency of
PTH-(1–34) was 12.7-fold greater in SaOS-2 cells than in HKRK-B28
cells (0.15 ± 0.01 vs. 1.9 ± 0.5
nM, respectively). The reasons for these
differences in the relative potencies of PTH-(1–34) and PTH-(1–14) in
the two cell lines are not clear, but we previously observed similar
reductions in the potencies of other PTH-(1–14) analogs relative to
PTH-(1–34) in ROS 17/2.8 cells, a rat osteoblastic cell line in which
moderate levels of PTH-1 receptors are expressed (80,000/cell)
(11). We also note that activity of native PTH-(1–14) was
barely detectable in SaOS-2 cells (Fig. 4).
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and Table
2). As in the cAMP assays, the most
potent amino-terminal peptide analog in the phospholipase C assays was
[Ala3,12,Gln10,Har11,Trp14]PTH-(1–14).
This peptide mediated a maximum response comparable with that of
PTH-(1–34), although the EC50 for this response
was much higher than that of PTH-(1–34) (EC50 =
11 ± 2 and 20 ± 4 nM, respectively).
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). The apparent binding affinity of
[Ala3, 10,
12,Arg11,Trp14]PTH-(1–14)
was 4-fold lower (IC50 = 350 ± 10
µM; P < 0.001; Table 2). The binding of
native PTH-(1–14) as well as that of the modified PTH-(1–11) analogs
were still too weak to be detected in HKRK-B28 cells (Table 2).
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, [Ala3,12,Gln10,Har11,Trp14]PTH-(1–14)
fully stimulated cAMP accumulation in COS-7 cells expressing
hP1Rc-delNt; the EC50 for this response
(0.25 ± 0.09 µM) was 170-fold lower than that
observed here for PTH-(1–34) (EC50 = 43 ±
14 µM; P = 0.01) and 3 log orders lower
than that observed previously for native PTH-(1–14) (see above). The
potency of
[Ala3,12,Gln10,Har11,Trp14]PTH-(1–14)
on hP1Rc-delNt was comparable to its potency measured on hP1Rc-WT
expressed in COS-7 cells (data not shown) as well as in HKRK-B28 cells
(Table 2). These results demonstrate that the homoarginine and other
activity-enhancing substitutions of the modified analog must mediate
their effects through the juxtamembrane region of the PTH receptor.
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, individual alanine mutations at
Phe184 and Leu187 in the
intact PTH-1 receptor strongly impaired the receptor’s responsiveness
to
[Ala3,Gln10,Har11]PTH-(1–11).
Thus, mutation of either of these receptor residues interferes with
effective interaction with the PTH-(1–11) analog.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial investigations at position 11 revealed new substitutions,
in particular homoarginine, that enhanced potency in PTH-(1–14) and
PTH-(1–11) analogs. Although we previously found that arginine and
lysine substitutions at position 11 in PTH-(1–14) enhanced peptide
activity (11), our current data showed that simply the
presence of a basic side-chain group is not sufficient for the
enhancing effect, as both [His11]PTH-(1–14)
(11) and [Orn11]PTH-(1–14) (Fig. 1) were substantially less potent than native PTH-(1–14). The data
suggest a beneficial effect of side-chains with aromatic
character/polarizability, as arginine-11-PTH-(1–14) was more potent
than citruline-11-PTH-(1–14), and homophenylalanine-11-PTH-(1–14) was
more potent than norleucine-11-PTH-(1–14) (Fig. 1). The data also
suggest that longer side-chains at position 11 are better than shorter
ones, as peptides with homoarginine or homophenylalanine at this
position were more active than their respective counterpart peptides
containing one less methylene unit in the position 11 side-chain, and
the lysine-11 analog was more active than the ornithine-11 analog.
The homoarginine-11 substitution could be combined with several of the
other activity-enhancing modifications that we recently described
(Ala3, Gln10,
Ala12, Trp14) to yield the
most potent PTH-(1–14) and PTH-(1–11) peptides identified to date.
These new homoarginine-containing peptides induced maximum or
near-maximum cAMP responses in heterologous cells expressing high
levels of PTH-1 receptors (e.g. HKRK-B28 cells) as well as
in SaOS-2 cells, a human osteoblast-like cell line with much lower
levels of endogenous PTH-1 receptors. The modified PTH-(1–14) and
PTH-(1–11) peptides, but not the unmodified peptide fragments, also
stimulated inositol phosphate production in COS-7 cells transfected
with the human PTH-1 receptor. The specificity of the modified peptides
for the PTH-1 receptor was preserved, as no signaling response was
detected in untransfected COS-7 or LLC-PK1 cells
(data not shown). Furthermore, the improvements in signaling potency
provided by the substitutions were accompanied by approximately
parallel improvements in apparent PTH-1 receptor binding affinity, as
seen in the capacities of the modified PTH-(1–14) analogs to inhibit
the binding of [125I]PTH-(3–34) to HKRK-B28
cells. The improved binding affinities of these analogs indicate that
at least some of the enhancement in signaling potency provided by the
modifications arises from gains in binding affinity, as opposed to pure
gains in signaling capability. The apparent binding affinities that we
observed for the PTH-(1–14) analogs in HKRK-B28 cells were still
considerably weaker than the corresponding cAMP signaling potencies
[by nearly 800-fold for
[Ala3,12,Gln10,Har11,-
Trp14]PTH-(1–14); Table 2]; such large
discrepancies suggest that the modifications have greater effects on
the intrinsic signaling capacities of the analogs than on their binding
affinities. It is important to note, however, that our binding studies
were performed under heterologous conditions, in that a relatively
unmodified PTH-(3–34) analog was used as a radioligand, and such
conditions may not yield true receptor binding affinities
(23). In any case, we infer from our binding studies that
the substitutions in the PTH-(1–14) analogs improve receptor binding
affinity, and that these effects on binding contribute to some of the
observed effects on signaling potency.
At present, our data do not allow us to determine whether the
substitutions in the amino-terminal PTH peptides stabilize or induce a
more favorable bioactive conformation in the ligand (an intramolecular
effect) or make direct and energetically beneficial contacts with the
receptor (an intermolecular effect). With regard to the latter
possibility, we used the new peptides as functional probes of receptor
interaction sites for agonist ligands. That rather substantial
structural changes to certain amino acid side-chains in the peptide
resulted in considerable improvements in potency (e.g.
30-fold for leucine-11
homoarginine) suggests that the introduced
amino acids may use contact sites in the receptor that are not used by
native PTH residues. Thus, the agonist-binding pocket of the PTH
receptor may not be stringently selective for the native hormone
sequence, but, in fact, may contain ancillary sites that can be
accessed for substantial gains in potency. The fact that the analogs
maintained full potency on the truncated receptor (which lacks most of
the amino- terminal extracellular domain) indicates that they exert
their effects through the juxtamembrane region of the receptor. The
recent cross-linking studies performed with the PTH-(1–34) analog
modified at lysine-13 indicate that the side-chain at this position of
PTH has the potential to contact the receptor near the boundary of the
amino-terminal extracellular domain and the juxtamembrane region,
specifically at arginine-186 (19). In our present study we
show that the presence of Phe184 and
Leu187 in this same receptor region is important
for mediating an agonist response to a ligand analog as short as
PTH-(1–11). The combined functional and cross-linking studies thus
suggest that there is at least proximity between the amino-terminal
portion of PTH agonist ligands and residues in the (184–187) region of
the receptor. Whether any of the side-chain modifications that we have
introduced into the N-terminal PTH analogs, such as homoarginine-11,
contact this portion of the receptor remains to be determined.
Overall, our data highlight the importance of ligand interactions to the juxtamembrane region of the PTH-1 receptor for inducing receptor activation. Certain native residues in the N-terminal portion of PTH ligands are clearly critical and may be required for efficient receptor activation, including valine-2, isoleucine-5, and methionine-8 (6, 8, 11, 16). Nevertheless, our data show that there is still a considerable degree of freedom in the interaction, such that the amino-terminal portion of PTH may be amenable to further modifications that enhance potency. Our current results show that by introducing extended side-chains with polarizable functional groups (guanidine, aromatic rings) at position 11, the functionality of short amino-terminal PTH peptides can be enhanced. These shorter length peptides could potentially offer advantages in terms of designing new PTH-based therapies for osteoporosis, and their inherently simpler structures render them useful as probes of the PTH-PTH receptor interaction mechanism. Such studies should provide new constraints for the ongoing efforts to model the ligand/receptor complex (8, 24) and thus should provide new clues for the development of even smaller peptide or nonpeptide PTH-1 receptor agonists. Indeed, our current findings lend support to the concept that it should be possible for a relatively low molecular weight compound to activate fully the PTH-1 receptor by interacting solely with its juxtamembrane region.
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Footnotes |
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Received December 19, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Shown are data (mean ± SEM) combined from five
separate experiments, each performed in duplicate.