The (1–14) 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent mutagenesis and cross-linking studies suggest that residues in the carboxyl-terminal portion of PTH(1–34) 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(1–14) and a truncated rat PTH-1 receptor (r{Delta}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 r{Delta}Nt, 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 r{Delta}Nt 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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(1–34), the major determinants of receptor-binding affinity reside within residues 15–34 (6, 7, 8, 9), and those of receptor activation lie within the highly conserved amino-terminal portion. Residues 1–6 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(1–27) 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 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTH(1–14) 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(1–15) 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. 1AGo, 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. 1AGo). 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. 1BGo).



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Figure 1. cAMP-Stimulating Activity of PTH Fragments in LLC-PK1 Cells

A, Rat PTH(1–34) 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(1–34) or rPTH(1–14), and intracellular cAMP was measured. Shown are data (mean ± SEM) from a single representative experiment performed in duplicate.

 
To identify residues in the PTH(1–14) fragment that play a role in activating the adenylyl cyclase-signaling pathway, we employed an alanine-scanning approach. Thirteen different alanine-substituted rat PTH(1–14) analogs were synthesized and tested for their ability to stimulate cAMP formation in HKRK-B7 cells (Fig. 2Go). 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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Figure 2. Alanine Scan of PTH(1–14)

HKRK-B7 cells were treated with 100 µM of one of 14 different rPTH(1–14) 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(1–34) at 0.1 µM was 254 ± 16 pmol/well.

 
PTH(1–14) Action in COS-7 Cells
To investigate the portions of the receptor required for PTH(1–14)-mediated signaling, we used a truncated rat PTH-1 receptor (r{Delta}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), r{Delta}Nt is predicted to contain only a short amino-terminal extracellular segment consisting of residues 23–26 joined to residues 182–190 (Fig. 3BGo). 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. 3CGo). Both peptides also stimulated cAMP formation with r{Delta}Nt, but whereas PTH(1–14) was equipotent with rWT-HA and r{Delta}Nt, PTH(1–34) was 2 orders of magnitude weaker with r{Delta}Nt than it was with rWT-HA (compare panels C and D of Fig. 3Go).



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Figure 3. PTH Responses of Intact and Truncated PTH-1 Receptors in COS-7 Cells

Shown at the top are schematics of the intact (A) and truncated (B) rat PTH-1 receptors used for transient transfection of COS-7 cells, and subsequent cAMP response assays. The conserved extracellular cysteine residues are depicted as open circles and numbered according to sequence position, and the nine amino acids of the epitope tag (HA) in rWT-HA are shaded. The tics at residue 26 and 181 indicate the endpoints of the deletion in r{Delta}Nt. Based on the predicted signal peptide cleavage site at Ala-22, residues 23–25 in r{Delta}Nt are joined to residue 182. The cAMP responses of COS-7 cells expressing the intact receptor (C) and r{Delta}Nt (D) to rPTH(1–34) (•) or rPTH(1–14) ({Delta}) are also shown. The graphs show combined data (mean ± SEM) from five separate experiments, each performed in duplicate.

 
The finding that the potency of PTH(1–14) was nearly equivalent with r{Delta}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{Delta}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{Delta}Nt-HA, exhibited signaling responses to PTH(1–14) that were equivalent to those seen for r{Delta}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(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. 4Go, A and B, the cAMP activity profile obtained with these analogs and r{Delta}Nt mirrored that obtained with rWT-HA (Fig. 4Go, A and B).



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Figure 4. Alanine Scan of PTH(1–14) with Intact and Truncated PTH Receptors

COS-7 cells transiently transfected with rWT-HA (A) or r{Delta}Nt (B) were treated with 100 µM of native rat PTH(1–14) or 100 µM of a rPTH(1–14) 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.

 
Specificity of Truncated Ligands and PTH Receptors
We examined whether PTH(1–14) and r{Delta}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. 5CGo). Cells expressing r{Delta}Nt 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. 5BGo). 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{Delta}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(1–34), 0.1 µM; rPTH(1–27), 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.

 
Inhibition of PTH(1–34)
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(1–14) on COS-7 cells expressing either rWT-HA or r{Delta}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. 6Go 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 r{Delta}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(1–14)

COS-7 cells transfected with rWT-HA (A) or r{Delta}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(1–14) 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(1–34) and PTH(1–14) 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.

 
PTH(1–14) as a Probe of Mutant Receptors
We tested the possibility that PTH(1–14) could be used as a functional probe for analyzing receptor mutations that alter interaction with PTH(1–34) 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(1–34). 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(1–14), whereas the Arg186 -> Ala mutation abolished PTH(1–14) activity (Fig. 7Go).



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Figure 7. PTH(1–14) as a Probe of Receptor Mutations

COS-7 cells were transfected with either the rWT-HA or with mutants of rWT-HA that had alanine point mutations at sites near the receptor’s amino terminus (Thr33 -> Ala and Gln37 -> Ala) or near the extracellular boundary of transmembrane helix 1 (Arg186 -> Ala) and were evaluated for responsiveness to PTH(1–14) (100 µM). Each of the three mutations caused a modest impairment in the binding of radiolabeled PTH(1–34) without detectable effects on PTH(1–27). As indicated, only the Arg186 -> Ala mutation impaired the cAMP-signaling response to PTH(1–14). Shown are the cAMP responses expressed as a percent of the response observed in control cells expressing rWT-HA and treated with PTH(1–34) (10 nM), which were included in each experiment. Shown are combined data (mean ± SEM) from two experiments each performed in duplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we show that the PTH(1–14) 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(1–34) than heretofore appreciated can induce receptor activation. It is notable that we could detect bioactivity with PTH(1–14) whereas previous studies on similar PTH or PTHrP fragments, such as PTH(1–12), 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(1–14) were inactive (Fig. 1Go), 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 r{Delta}Nt, even though ligand binding is too weak to measure (35).

In contrast to the similar potency that PTH(1–14) exhibited with r{Delta}Nt and rWT-HA, PTH(1–34) was 100-fold weaker with r{Delta}Nt than it was with the intact receptor (Fig. 3Go, 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 r{Delta}Nt (Fig. 3DGo) 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. 6BGo) 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 r{Delta}Nt (e.g. residues 182–190). Arginine-186 is important for PTH(1–14) activity (Fig. 7Go) 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. 1BGo). 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 r{Delta}Nt 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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(1–14) 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(1–34) control peptide, [Nle8,21,Tyr34]rPTH-(1–34)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(1–14) 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(1–34), 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 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 r{Delta}Nt, was originally referred to as r{Delta}E1-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. 3BGo). A similar truncated receptor with an amino-terminal epitope tag, r{Delta}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{Delta}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(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.


    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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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