Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand*

Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by re-placing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1–9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane do-main-1. Expression of this construct, hP1Rc-Tether(1–9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 3 Arg yielded hP1Rc-[Arg 11 ]Tether(1–11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg 11 ]Tether(1–11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information con-cerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane in COS-7 Cells— The pCDNA-1-based plasmid encoding the intact hPTH-1 receptor (13) (hP1Rc-WT) was used as a starting plasmid for mutagenesis. The truncated human PTH-1 receptor (hP1R-delNt) is deleted for residues 24 to 181 and was constructed from hP1Rc-WT by oligonucleotide-directed mutagenesis (14). Signal peptidase cleavage of this receptor is predicted to occur between Ala-22 and Tyr-23 (15) and, thus, leave

Human parathyroid hormone (hPTH) 1 is an 84-amino acid polypeptide that binds to a class B G protein-coupled receptor, the PTH-1 receptor, and thereby plays a vital role in regulating the extracellular concentrations of ionized calcium (1). The PTH-1 receptor also mediates the actions of PTH-related peptide, a large (ϳ140 amino acids) polypeptide ligand that plays a critical role in the developmental of several organs, particularly the skeleton (1). PTH has potent anabolic effects on bone in humans (2) and, thus, is a potential therapy for metabolic bone diseases, such as osteoporosis (3). Structure-activity analyses of PTH have shown that the 1-34 fragment is sufficient for full biological activity (4). Within the PTH(1-34) peptide, the N-terminal residues are the most critical for receptor activation, whereas the C-terminal residues are more important for determining receptor binding affinity (4,5).

Peptides-The
PTH Receptor Mutagenesis and Expression in COS-7 Cells-The pCDNA-1-based plasmid encoding the intact hPTH-1 receptor (13) (hP1Rc-WT) was used as a starting plasmid for mutagenesis. The truncated human PTH-1 receptor (hP1R-delNt) is deleted for residues 24 to 181 and was constructed from hP1Rc-WT by oligonucleotidedirected mutagenesis (14). Signal peptidase cleavage of this receptor is predicted to occur between Ala-22 and Tyr-23 (15) and, thus, leave * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
cAMP Stimulation-Measurements of cAMP formation in COS-7 cells were performed in 24-well plates 4 days after transfection (11). The cells were rinsed with 0.5 ml of binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 5% heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH 7.7 with HCl) and treated with 200 l of IBMX buffer (Dulbecco's modified Eagle's medium containing 2 mM 3-isobutyl-1-methylxanthine, 1 mg/ml bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4) and 100 l of binding buffer (basal) or binding buffer containing varying amounts of a PTH peptide analog (total volume ϭ 300 l). The cells were then incubated at room temperature for 1 h (or less for the time course experiment of Fig. 4), and the IBMX-containing buffer was removed. The cells were immediately frozen on crushed dry ice, and then 0.5 ml of 50 mM HCl was added, and the cells were refrozen on dry ice. The cAMP content of the thawed and diluted lysate was determined by radioimmunoassay.
Data Calculation-Calculations were performed using Microsoft Excel. For some experiments, a four-parameter nonlinear regression equation was used to fit curves to the cAMP dose-response data and to obtain the corresponding EC 50 values (16). The statistical significance between two data sets was determined using a one-tailed Student's t test assuming unequal variances for the two sets.

RESULTS
To construct PTH receptors having the PTH ligand sequence covalently "tethered" to the body of the receptor, we utilized as a starting scaffold a human PTH-1 receptor analog that is deleted for most of the extracellular N-terminal domain, hP1Rc-delNt (11). This deletion mutant receptor retains the native signal sequence, as do each of the subsequent tethered receptor constructs, such that signal peptidase cleavage between the Ala-22 and Tyr-23 peptide bond is predicted to generate the mature receptor mutant with an N-terminal tyrosine residue (15). In hP1Rc-delNt, this tyrosine is joined directly to glutamate 182; in the tethered receptors, the PTH segment, linked C-terminally to a tetraglycine linker, is inserted between the N-terminal tryrosine and glutamate 182 (Fig. 1). The N-terminal tyrosine was not expected to be a major detriment to the potential signaling activity induced by the tethered PTH segment because [Tyr Ϫ1 ]rPTH(1-14)NH 2 was 50% as active as native PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) in stimulating a cAMP response in cells expressing the P1Rc. 3 The basal and ligand-stimulated signaling properties of the tethered PTH-1 receptors in transiently transfected COS-7 cells are shown in Fig. 2. In the absence of added agonist, cells expressing hP1R-Tether(1-9) exhibited basal cAMP levels that were 10-fold higher than those observed with hP1Rc-WT or hP1Rc-delNt ( Fig. 2A). Extension of the ligand chain by one or two residues yielded hP1Rc-Tether(1-10) and hP1Rc-Tether(1-11), which produced moderate, but statistically significant (p Ͻ 0.005), improvements in the levels of basal cAMP signaling relative to hP1Rc-Tether(1-9). Basal cAMP signaling was increased substantially by the replacement of the native leucine at position 11 of hP1R-Tether(1-11) with arginine; we recently found that this same substitution enhances cAMP potency in short N-terminal PTH(1-14) analog peptides. 2 Each of the tethered hP1Rc constructs responded to a 1 M dose of exogenous PTH(1-34) analog to a similar extent (Fig. 2B).
To further evaluate the ability of these tethered receptors to respond to exogenous ligands, we performed dose-response analyses using rPTH(1-34), the Q-PTH(1-34) analog, 2 and the N-terminal PTH(1-14) fragment (11). The receptor hP1Rc-Tether(1-9) exhibited agonist responses to these ligands that were much the same as those seen with hP1Rc-delNT (Fig. 3, B and C). With hP1Rc-[Arg 11 ]Tether(1-11), only a weak stimulation of cAMP accumulation could be discerned with the fulllength peptides, and no increase in cAMP accumulation above the already high basal level was detected for the shorter PTH(1-14) peptide (Fig. 3D).
The rate of cAMP accumulation induced by hP1Rc-  1. Design of tethered PTH-1 receptors. Shown are schematics of the PTH-1 receptor constructs analyzed in this study. The tethered receptors (center) have residues 24 to 181 of the human PTH-1 receptor replaced by the N-terminal residues of rat PTH or the [Arg 11 ]PTH analog, as indicated by the boxed sequences. These receptor constructs thus retain the native hPTH-1 receptor signal sequence (Met-1 to Tyr-23) such that the predicted signal peptidase cleavage at the Ala-22/Tyr-23 peptide bond generates the mature tethered receptor (shown) having an N-terminal tyrosine followed by the PTH ligand sequence (N-terminal to C-terminal) joined C-terminally via a tetraglycine linker to Glu-182 of the receptor (residue position numbers corresponding to the wild type PTH-1 receptor sequence). In hP1Rc-delNt, which was used to construct the tethered receptors and serves as an experimental control, Tyr-23 is joined directly to Glu-182. The relative positions of Glu-182 and Tyr-23 in each receptor construct are depicted as a solid diamond and a tic mark, respectively. (Fig. 5). The DNA-dependence of these responses were parallel to those seen with a constitutively active PTH-1 receptor that contained the His-223 3 Arg point mutation at the cytoplasmic end of transmembrane helix 2 (17). At each equivalent DNA dose, the basal cAMP response observed with hP1Rc-[Arg 11 ]Tether(1-11) was approximately twice that observed for hP1Rc-H223R (range ϭ 1.6 -2.3-fold).
The finding that the Leu-11 3 Arg substitution substantially improved the signaling activity of both the tethered receptor construct and a synthetic PTH(1-14) peptide 2 prompted us to investigate whether other similarities existed between the structure-activity profiles of free PTH peptides and the PTH portion of the tethered receptor. Thus, we performed an alanine scan analysis of the PTH residues 2-9 of hP1Rc-[Arg 11 ]Tether(1-11), because these amino acid positions correspond to the most critical functional sites in PTH(1-14) (11). As illustrated in Fig. 6A, position-specific effects of these alanine substitutions on the basal cAMP-signaling activity of hP1Rc-[Arg 11 ]Tether(1-11) were observed. The alanine substitutions of glutamine 6 and histidine 9 had relatively mild effects on basal signaling. These results stand in contrast to the severe effects that alanine substitutions at positions 6 and 9 had on PTH(1-14) peptide activity (11). Alanine substitutions of glutamate 4 and leucine 7 had intermediate effects on the basal signaling of hP1Rc-[Arg 11 ] Tether(1-11). The most severe reductions in basal activity occurred with the alanine substi-tutions of valine 2, isoleucine 5, and methionine 8. These strong reductions in activity paralleled the effects that substitutions at the corresponding positions in PTH(1-14) had on cAMPsignaling activity (11). 2 Each of the alanine-substituted tethered receptors mediated a response to exogenous PTH(1-34) analog (1 M) that was comparable with that observed with the unmodified hP1Rc-[Arg 11 ]Tether(1-11) control receptor (Fig. 6B).

DISCUSSION
This study describes a series of novel tethered PTH ligand/ PTH-1 receptor constructs in which the N-terminal residues of the PTH ligand are fused to the body of the heptahelical G protein-coupled human PTH-1 receptor. This approach was pursued as a means to potentially simplify the analysis of the interactions between PTH and its receptor. This interaction can be otherwise difficult to evaluate, given the high degrees of freedom inherent to a bimolecular system involving large diffusible proteins of uncertain three-dimensional structures. Each of the tethered receptors in our study exhibited elevated basal cAMP signaling levels, as compared with hP1Rc-WT, when transiently expressed in COS-7 cells. The highest constitutive activity occurred with hP1Rc-[Arg 11 ] Tether(1-11), for which the basal cAMP levels were 75% of the maximum cAMP response attained by PTH(1-34)-treated hP1Rc-WT. The basal signaling activities of the tethered receptors were dependent on the amount of DNA used for the COS-7 cell transfection and followed a time course that closely resembled that observed for the agonist-stimulated hP1Rc-WT. The basal signaling of hP1Rc-[Arg 11 ]Tether(1-11) resulted in approximately twice the level of intracellular cAMP that was obtained with a previously described constitutively active PTH-1 receptor, hP1Rc-H223R (17), when the two receptors were transfected using equal amounts of plasmid DNA. The ability of a small peptide derived from the activation domain of PTH to stimulate G protein coupling when tethered to the body of the PTH receptor bears direct similarity to the intramolecular mechanism of activation utilized by the protease-activated receptors, such as the well characterized thrombin receptor (18).
For the tethered PTH receptors of the present study, the native ligand sequences of PTH(1-9), PTH(1-10), and PTH(1-11) were weaker than the tethered PTH(1-11) sequence containing the Leu 11 3 Arg substitution, even though each of these tethered ligands was present at the same equimolar ratio, relative to the concentration of the membrane-embedded portion of the receptor. The level of expression of these receptors was likely to be comparable, given that each stimulated similar maximum levels of cAMP in response to high doses of an exogenous PTH(1-34) analog. There was not a simple correlation between PTH chain length and basal activity because hP1Rc-Tether(1-11) was statistically weaker than hP1Rc-Tether(1-10) (34 Ϯ 1 and 52 Ϯ 2 pmol/well, respectively, p Ͻ 0.001). The improved basal signaling of the Arg-11-containing tethered ligand is consistent with the favorable effect that this substitution had on the potency of PTH(1-11) and -(1-14) synthetic peptide analogs. 2 This observation supports the hypothesis that the tethered PTH ligands utilize the same contact points for activating the receptor as do exogenous PTH peptide ligands.
The reasons for the greater mutational tolerance that we observed for certain PTH residues in the context of hP1Rc-[Arg 11 ]Tether (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), in comparison to similarly substituted PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) peptides (e.g. Gln-6, Leu-7, and His-9), are not clear at present. It is possible that the free PTH peptide and the tethered ligand utilize slightly different modes of receptor interaction. It also possible that an alanine substitution has a different effect on the secondary structure of the ligand when the ligand is a free peptide, as compared with when it is in a tethered configuration. A third possibility is that the high effective molarity of the tethered ligand could allow for a discrimination between those residues that principally affect receptor signaling and those that principally affect ligand binding. Residues of the latter category would be critical for detecting activity in short diffusible peptides but not when the same ligand sequence is fixed to the receptor, whereas residues of the former category would be essential in both situations. The ability to discern such a structure-function relationship within short N-terminal PTH peptides has not been available previously, because the cAMP potency and efficacy of any given analog is inextricably linked to its affinity for the receptor (12). Further work is needed to determine the precise contribution that each residue of the tethered PTH ligand makes toward the constitutive signaling activity of the receptor.
The results presented in this report suggest that the tethered ligand system can offer new insights into the mechanism by which the N-terminal residues of PTH interact with the PTH-1 receptor and induce transmembrane signaling. The information from these and future studies on the tethered receptors could help to constrain the emerging three-dimensional models of the PTH/PTH-1 receptor complex. The dramatic minimization of the bioactive ligand sequence that is now possible due to the elimination of the need for high affinity binding should simplify such analyses and could lead to a better definition of the minimal pharmacophore required for PTH-1 receptor activation.