INTRODUCTION

The parathyroid hormone (PTH¹)-2 receptor, a recently identified PTH receptor subtype, responds fully to PTH but not at all to PTHrP (1). This ligand selectivity profile of the PTH-2 receptor is dramatically different from that of the PTH-1 receptor (PTH/PTHrP receptor) which elicits a robust increase in cAMP formation in response to either ligand. The amino acid sequences of the two receptors are 51% identical, and each is a member of the subfamily of G protein-coupled receptors that bind peptide hormones of intermediate size including calcitonin, secretin, glucagon, vasoactive intestinal peptide, and several other peptides (2), in addition to PTH and PTHrP. These receptors are characterized by a relatively large amino-terminal extracellular domain of 100-200 amino acids, which contains six highly conserved cysteine residues, a "core" region with seven hydrophobic transmembrane helices and connecting loops, and a carboxyl-terminal tail of 150-200 amino acids.

The molecular basis by which the peptide hormone receptors engage their respective receptors and trigger receptor activation is still largely unknown. This problem has been approached through strategies involving the construction of receptor chimeras and other types of receptor mutants (3-12). The data emerging from these studies suggest that multiple segments of the ligand and receptor contribute to the interaction. Recent studies with chimeric ligands acting on chimeric receptors suggest that the carboxyl-terminal portion of the ligand interacts with the amino-terminal extracellular domain, whereas the amino-terminal portion of the hormone interacts with the membrane-spanning/loop region of the receptor (8). However, as yet, there are only limited data on the specific amino acids, either in the ligand or in the receptor, that contribute to the interaction.

In other receptor systems, the availability of receptor subtypes that exhibit distinct pharmacological profiles for different ligands has facilitated the identification of receptor residues involved in ligand recognition (13). The pronounced difference in the ligand selectivity profiles of the PTH-1 and PTH-2 receptors suggested that these two receptors could be used in such an analysis, since the difference in selectivity can most easily be explained by structural differences in the receptors at sites (or a site) that are involved in ligand recognition or ligand-induced receptor activation. Further, it suggests that the two ligands differ at residues that interact with these divergent receptor residues. Recently, two such residues in PTH and PTHrP that can account for their altered selectivity for the PTH-2 receptor were identified: residue 23, Phe in PTHrP and Trp in PTH, modulates ligand binding (14); and residue 5, His in PTHrP and Ile in PTH, modulates ligand-induced receptor activation (14, 15). Thus, the weak binding of PTHrP to the PTH-2 receptor can be explained by the presence of Phe²³, and the weak signaling activity at this receptor can be explained by the histidine at position 5.

In the present paper we use a receptor chimera and mutagenesis approach to search for sites in the PTH-2 receptor that are involved in His⁵/Ile⁵ signaling selectivity. The results reveal two divergent amino acids in the membrane-spanning and extracellular loop portion of the receptor that contribute strongly to this effect.

EXPERIMENTAL PROCEDURES

The preparation and initial characterization of [Trp²³,Tyr³⁶]PTHrP-(1-36)amide and [Ile⁵,Trp²³,Tyr³⁶]PTHrP-(1-36)amide was described previously (14). Herein, these two peptides are referred to as [Trp²³]PTHrP-(1-36) and [Ile⁵,Trp²³]PTHrP-(1-36), respectively. These PTHrP analogs, and other peptides used in the study, were prepared by the biopolymer synthesis facility at Massachusetts General Hospital (Boston, MA), as were the DNA oligonucleotides used in receptor mutagenesis experiments. The PTH analog [Nle^8,21,Tyr³⁴]rPTH-(1-34)amide was radioiodinated by the chloramine-T procedure, and the product was purified by reverse phase high performance liquid chromatography (16). ¹²⁵I-Na (2,000 Ci/mmol) was purchased from NEN Life Science Products. Dulbecco's modified Eagle's medium, EGTA/trypsin, and concentrated antibiotic mixture (10,000 units/ml penicillin G and 10 mg/ml streptomycin) were from Life Technologies, Inc.; fetal bovine serum was from HyClone Laboratories (Logan, UT). DNA modifying reagents were from United States Biochemical Corp. (Cleveland, OH) or New England Biolabs, Inc. (Beverly, MA).

The cDNAs encoding the human PTH-1 (PTH/PTHrP) receptor (17) and the human PTH-2 receptor (1) were carried on the expression vectors pcDNA-1 and pcDNAI/Amp (InVitrogen, San Diego, CA), respectively. The 1E2 and 2E1 receptor chimeras were constructed by utilizing the unique, naturally occurring EcoRI site in the PTH-2 receptor plasmid that overlaps codons 139/140. A matching EcoRI site was introduced into the human PTH-1 receptor by oligonucleotide-directed mutagenesis at codons 182/183 to generate the receptor plasmid pHK-FE. The mutagenic oligonucleotide used to make pHK-FE also changed Val¹⁸³ and Asp¹⁸⁵ to Phe and Glu, respectively, which correspond to the amino acid sequence of the PTH-2 receptor. The HK-FE and WT PTH-2 receptor plasmids were cleaved with BamHI, which cuts in the 5 polylinker region, and EcoRI, and then the appropriate EcoRI-BamHI DNA fragments were gel-purified and religated to yield the desired chimeras. The chimera 1E2 has residues 1-182 of the P1R joined to residues 140-550 of the P2R, and chimera 2E1 has residues 1-142 of the P2R joined to residues 186-593 of the P1R. All other cassette and point mutations were introduced by oligonucleotide-directed mutagenesis (18).

COS-7 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%), penicillin G (20 units/ml), and streptomycin (20 µg/ml) in a humidified atmosphere containing 5% CO₂. Cells were transfected in 24-well plates using plasmid DNA (200 ng/well) that was purified by cesium chloride/ethidium bromide gradient centrifugation, except for the initial screening of the cassette mutants, in which phenol-extracted miniprep DNA was used. This miniprep DNA was quantified by ethidium bromide staining of agarose gels, and was transfected at a concentration of 100 ng/well. 48 h after transfection, the cell medium was replenished and the plates were shifted to a 33 °C humidified incubator for an additional 24-48 h, by which time the cell density reached 500,000 ± 100,000 cells/well. This shift to a lower temperature resulted in a general 10-50% increase in the number of receptors on the cell surface, as compared with cells maintained at 37 °C² as has been found for other G protein-coupled receptors (19). The cells were then used for binding and cAMP stimulation assays.

Binding reactions were performed as described previously (18). Each well (final volume = 300 µl) contained 26 fmol of ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)NH₂ (100,000 cpm) and various amounts (0.4-300 pmol) of unlabeled competitor ligand; peptides were diluted in 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 fetal bovine serum). Incubations were at room temperature for 2 h, except for experiments performed for Scatchard analysis, which were performed at 4 °C for 6 h. At the end of the binding reactions the cells were rinsed 3 times with 0.5 ml of binding buffer, lysed with 0.5 ml of 5 M NaOH, and the entire lysate was counted. Nonspecific binding of tracer (NSB), determined in wells containing 1 µM [Nle^8,21,Tyr³⁴]rPTH-(1-34)NH₂, was 1-1.5% of total counts added. Maximum specific binding (B0) was calculated as the total radioactivity bound to cells in the absence of unlabeled ligand minus NSB. IC₅₀ values (dose of competing ligand that resulted in 50% inhibition of ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)NH₂ binding) were determined from plots of log(B/B0  B) versus log[competitor]. Cell surface receptor numbers were estimated from Scatchard analyses of homologous competition binding studies that were performed with ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)NH₂ (26 fmol/well) and varying amounts (1.2-300 pmol) of the same unlabeled ligand. Calculations of the number of receptors per cell assumed a single class of binding sites and a transfection efficiency of 20% (6, 20).

Transfected COS-7 cells were rinsed with 500 µl of binding buffer, and 200 µl 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 µl of binding buffer or binding buffer with various amounts of peptide added. The plates were incubated for 60 min at room temperature; the buffer was then withdrawn and the cells were lysed by adding 0.5 ml of 50 mM HCl and freezing. The diluted lysate (1:30 in distilled H₂0) was analyzed for cAMP content by radioimmunoassay. For the initial screening of the mutants, we compared the cAMP response of each receptor to a maximum dose (1 µM) of [Trp²³]PTHrP-(1-36) to its response to the nonselective analog [Ile⁵,Trp²³]PTHrP-(1-36) also at 1 µM. Dose-response analyses yielded EC₅₀ values (ligand dose resulting in 50% of maximum response (E_max) attained by that ligand), which were calculated from plots of log(E/E_max  E) versus log[ligand], where E is the cAMP response measured at the corresponding dose of ligand (6).

RESULTS

The difference in the ligand selectivities of the PTH-1 receptor and the PTH-2 receptor can be seen in the cAMP response profiles shown in Fig. 1, panels A and B. The PTH-1 receptor responded fully and equally to both [Ile⁵, Trp²³]PTHrP-(1-36) ([Ile⁵,Trp²³,Tyr³⁶]PTHrP-(1-36)amide) and [Trp²³]PTHrP-(1-36) ([Trp²³,Tyr³⁶]PTHrP-(1-36)amide), which has histidine at position 5. In contrast, the PTH-2 receptor responded fully to [Ile⁵,Trp²³]PTHrP-(1-36) but not at all to [Trp²³]PTHrP-(1-36). The ligand selectivity of the PTH-2 receptor is not due to a difference in binding affinities because the two analogs exhibited comparable potencies in their ability to inhibit the binding ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)amide (Fig. 1F). The high apparent binding affinity that the two PTHrP analogs displayed for the PTH-2 receptor in these experiments is due primarily to the Phe²³  Trp modification; this substitution of a PTHrP residue by the corresponding PTH residue markedly enhances binding potency at the PTH-2 receptor without affecting cAMP signaling (14). A small improvement in binding potency was also seen for the His⁵  Ile modification, however, this improvement was not receptor specific (Fig. 1, E and F) and was much smaller in magnitude than the effect of the substitution on PTH-2 receptor signaling.

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To localize the region of the PTH-2 receptor involved in His⁵/Ile⁵ signaling selectivity, we constructed a pair of chimeras in which the amino-terminal extracellular domains of the human PTH-1 and PTH-2 receptors were reciprocally interchanged and tested the chimeras in cAMP stimulation assays for responsiveness to [Trp²³]PTHrP-(1-36) and to the nondiscriminating control peptide [Ile⁵,Trp²³]PTHrP-(1-36). The 1E2 receptor chimera, which has the amino-terminal extracellular domain of the PTH-1 receptor connected (via an EcoRI site) to the mid- and carboxyl-terminal region of the PTH-2 receptor, discriminated between the two ligands (Fig. 1C), whereas the reciprocal chimera 2E1 responded fully to each ligand (Fig. 1D). These results indicated that the membrane-spanning and loop portion of the receptor determines His⁵/Ile⁵ signaling selectivity.

To further localize the sites involved in residue 5 selectivity, we replaced most of the divergent residues in the membrane-spanning helices and extracellular connecting loops of the PTH-2 receptor with the corresponding residues of the PTH-1 receptor. As shown in Fig. 2, these residues were replaced either by cassette substitution or, for two of the sites (Ala³²⁵ and Gly³²⁷), by single residue point mutation. All but 3 of the 21 mutant receptors were functional and adequately expressed on COS-7 cells, as judged by the cAMP response to [Ile⁵,Trp²³]PTHrP-(1-36) and the binding of radioiodinated [Nle^8,21,Tyr³⁴]rPTH-(1-34)amide (Table I). The three cassette mutants showing poor cAMP responsiveness and little or no PTH binding, P2R-Csst#2, P2R-Csst#7 and P2R-Csst#18, were possibly not expressed on the cell surface and were considered uninformative. Of the functional mutant receptors, three displayed increased responsiveness to [Trp²³]PTHrP-(1-36). These three cassettes are predicted to be located in the amino-terminal portion of extracellular loop 1 (P2R-Csst#5), the extracellular end of helix 3 (P2R-Csst#8), and at the COOH-terminal end of extracellular loop 2 (P2R-Csst#13) (Fig. 2). For each of these three receptor mutants the maximum binding of ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)amide was elevated by a factor of 1.8-3.2, in comparison to the binding observed for the WT PTH-2 receptor (Table I). This increase in maximum binding of radiolabeled PTH-(1-34) may indicate increased surface expression, enhanced PTH-(1-34) binding affinity, or both. It is unlikely, however, that such effects on surface expression or PTH-(1-34) binding affinity are the basis for the altered cAMP responsiveness to [Trp²³]PTHrP-(1-36), because other mutants, such as P2R-Csst#16 and GV-327, caused comparable increases in radioligand-binding capacity without changing [Trp²³]PTHrP-(1-36) signaling selectivity (Table I). We therefore focused on the receptor regions defined by cassette mutations 5, 8, and 13. 

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Table I. Localization of PTHrP-signaling determinants in the PTH-2 receptor WT and mutant PTH receptors were expressed in COS-7 cells and evaluated for binding 125I-rPTH-(1-34) analog, and for intracellular cAMP levels, in the absence of added ligand (basal) or presence of [Trp23,Tyr36]hPTHrP-(1-36)NH2 (PTHrP) or [Ile5,Trp23,Tyr36]hPTHrP(1-36)NH2 ([Ile5]PTHrP), each at a dose of 1 µM. The top portion of the table shows data from the 19 cassette mutations and 2 point mutations used in initial screening studies. The lower half shows the effect of point mutations made within regions defined by cassettes 5, 8, and 13. The far right column shows the ratio of the net responses to the two analogs. Data are the means ± S.E. of two experiments, each performed in duplicate. For some mutants, PTHrP responsiveness was not determined because of probable defects in expression (ND), or because the response to PTHrP was less than the basal cAMP level (< bsl). Receptor Maximum binding 125I-PTH-(1-34) cAMP Basal [Ile5]PTHrP PTHrP PTHrP/[Ile5]PTHrP % P2R-WT pmol/well % P2R-WT 100  ± 1 6.1  ± 0.6 55.6  ± 1.8 6.9  ± 0.8 1.8  ± 1.0 P1R-WT 538  ± 57 4.4  ± 1.1 148.5  ± 32.3 142.6  ± 35.2 95.3  ± 8.6 P2R cassette mutation   1 39  ± 4 3.9  ± 1.1 51.5  ± 2.5 3.1  ± 0.8 < bsl   2 11  ± 3 1.9  ± 0.4 1.6  ± 0.4 2.1  ± 0.5 ND   3 66  ± 2 3.8  ± 0.4 48.8  ± 5.6 4.8  ± 0.6 2.4  ± 1.0   4 122  ± 4 4.8  ± 0.4 39.4  ± 4.6 5.9  ± 0.6 3.8  ± 1.8   5 317  ± 25 4.5  ± 0.4 80.9  ± 8.3 36.0  ± 4.0 40.5  ± 1.5   6 21  ± 5 3.5  ± 0.4 32.5  ± 8.9 5.5  ± 0.3 9.2  ± 2.7   7 0.0  ± 0.7 2.2  ± 0.6 2.5  ± 0.8 2.1  ± 0.5 ND   8 181  ± 9 5.3  ± 0.6 54.9  ± 6.3 69.2  ± 8.7 126.9  ± 4.5   9 96  ± 9 9.6  ± 1.1 90.8  ± 20.1 22.9  ± 4.1 16.3  ± 1.0   10 92  ± 6 4.8  ± 0.6 81.2  ± 3.7 4.5  ± 1.0 < bsl   11 93  ± 10 4.0  ± 0.4 72.9  ± 8.9 11.9  ± 0.3 11.6  ± 0.5   12 130  ± 9 3.3  ± 0.7 99.1  ± 9.9 16.3  ± 1.4 13.5  ± 0.4   13 221  ± 14 1.6  ± 0.4 64.9  ± 23.5 37.9  ± 13.3 57.3  ± 0.9   14 91  ± 4 4.3  ± 0.4 59.6  ± 9.4 3.9  ± 0.5 < bsl   15 157  ± 7 11.5  ± 0.8 90.3  ± 9.5 30.4  ± 2.4 24.3  ± 0.9   16 203  ± 10 4.4  ± 0.3 64.2  ± 7.9 7.7  ± 0.4 6.1  ± 0.6   17 129  ± 12 3.4  ± 0.7 75.2  ± 11.7 5.4  ± 0.2 3.1  ± 0.7   18 27  ± 2 1.7  ± 0.2 2.2  ± 0.3 1.8  ± 0.2 ND   19 95  ± 2 4.1  ± 0.3 51.9  ± 5.9 4.4  ± 0.2 1.0  ± 0.6   AS-325 89  ± 3 4.0  ± 0.7 39.3  ± 3.1 3.6  ± 0.5 < bsl   GV-327 191  ± 11 7.6  ± 1.4 70.6  ± 15.1 11.0  ± 2.0 5.2  ± 1.2 P2R point mutation parent cassette   RA-199 5 104  ± 25 7.5  ± 0.9 111.2  ± 22.3 9.7  ± 0.1 2.8  ± 1.1   VL-201 5 114  ± 5 3.4  ± 0.6 57.5  ± 21.1 6.3  ± 1.5 5.3  ± 0.9   HY-202 5 53  ± 2 5.0  ± 1.4 49.8  ± 9.3 4.2  ± 0.2 < bsl   AS-203 5 105  ± 5 6.6  ± 1.0 62.3  ± 16.5 7.4  ± 0.7 2.5  ± 1.5   HG-204 5 128  ± 3 4.6  ± 0.9 46.1  ± 3.2 7.4  ± 0.7 6.7  ± 0.4   IA-205 5 78  ± 4 5.9  ± 0.4 62.2  ± 12.8 6.4  ± 0.2 1.5  ± 1.0   GT-206 5 84  ± 7 3.7  ± 0.3 37.7  ± 5.6 4.6  ± 0.8 3.3  ± 1.9   VL-207 5 4.6  ± 2.8 1.5  ± 0.5 1.2  ± 0.4 1.5  ± 0.6 ND   KD-208 5 8.6  ± 2.7 1.3  ± 0.4 1.9  ± 0.4 1.9  ± 0.5 ND   KR-237 8 37  ± 4 5.3  ± 0.2 24.2  ± 4.4 4.5  ± 0.4 < bsl   IV-238 8 110  ± 4 4.3  ± 0.1 39.6  ± 6.5 5.0  ± 0.4 1.7  ± 0.9   VT-241 8 75  ± 5 5.0  ± 0.8 82.4  ± 9.5 4.6  ± 0.2 < bsl   MF-242 8 95  ± 7 5.4  ± 0.3 68.7  ± 10.9 6.0  ± 0.3 0.8  ± 0.4   IL-244 8 176  ± 3 6.0  ± 0.7 37.9  ± 9.0 40.3  ± 12.9 98.2  ± 22.2   IK-314 13 96  ± 6 4.8  ± 0.2 51.6  ± 8.5 4.3  ± 0.5 < bsl   YI-318 13 261  ± 17 6.8  ± 3.3 92.4  ± 6.4 66.3  ± 1.0 69.5  ± 3.9   AV-321 13 119  ± 6 5.2  ± 0.5 64.7  ± 6.7 9.8  ± 0.4 7.9  ± 0.8

Replacement of each divergent residue in cassette region 5 by the corresponding residue of the PTH-1 receptor failed to identify a single residue affecting the cAMP response to [Trp²³]PTHrP-(1-36) (Fig. 3B and Table I). It is possible that two or more sites in this region cooperatively contribute to His⁵/Ile⁵ signaling selectivity; however, two of the mutations in this set, Val²⁰⁷  Leu (valine 207 changed to leucine, VL-207) and Lys²⁰⁸  Asp (KD-208) were poorly expressed, as indicated by their very low responses to [Ile⁵,Trp²³]PTHrP-(1-36) and minimal binding of ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)amide. Thus, the roles of these two residues could not be assessed. Within cassette region 8, one point mutation, Ile²⁴⁴  Leu (IL-244), resulted in a strong increase in responsiveness to [Trp²³]PTHrP-(1-36) (Fig. 3B). Within cassette region 13, one other substitution Tyr³¹⁸  Ile (YI-318), also enhanced responsiveness to the PTHrP analog. Dose response analysis of these two mutant receptors demonstrated that each mutation by itself could account for a substantial component of the signaling selectivity inherent to the PTH-2 receptor (Fig. 4). In fact, with the IL-244 receptor, the efficacy of [Trp²³]PTHrP-(1-36) was equal to, if not slightly greater than, that of [Ile⁵,Trp²³]PTHrP-(1-36) (Fig. 4B and Table II). The effect of the YI-318 mutation was not as pronounced, and with this mutant [Trp²³]PTHrP-(1-36) functioned as a partial agonist (Fig. 4C). These two point mutations did not affect the ability of either PTHrP analog to inhibit the binding of ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)amide (Fig. 4 D-F). Scatchard analyses indicated that the number of PTH-(1-34) binding sites on the surface of COS-7 cells transfected with either mutant receptor did not differ significantly from the number of binding sites on cells expressing the WT PTH-2 receptor (Table III).

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Table II. cAMP responses of WT and mutant PTH-1 and PTH-2 receptors Values are means ± S.E. of six to twelve experiments performed in COS-7 cells. The peptides tested were [Trp23,Tyr36]PTHrP-(1-36)NH2 ([Trp23]PTHrP-(1-36)) and [Ile5,Trp23,Tyr36]PTHrP-(1-36)NH2 ([Ile5,Trp23]PTHrP-(1-36)). The maximum cAMP responses (basal not subtracted) were determined at a peptide dose of 1 µM. The low cAMP response of the WT PTH-2 receptor to [Trp23]PTHrP-(1-36) precluded the determination of an EC50 value. Receptor basal cAMP [Trp23]PTHrP-(1-36) [Ile5,Trp23]PTHrP-(1-36) maximum EC50 maximum EC50 pmol/well nM pmol/well nM P2R-wt 18  ± 2 19  ± 3 134  ± 17 1.8  ± 0.4 P2R-IL-244 25  ± 4 135  ± 24 1.9  ± 0.5 99  ± 24 2.5  ± 1.1 P2R-YI-318 10  ± 2 84  ± 24 2.9  ± 0.9 122  ± 32 0.5  ± 0.1 P1R-wt 18  ± 4 356  ± 55 0.6  ± 0.1 287  ± 27 0.7  ± 0.2 P1R-LI-289 25  ± 8 327  ± 69 0.5  ± 0.1 288  ± 50 0.9  ± 0.3 P1R-IY-363 12  ± 3 112  ± 26 0.9  ± 0.4 103  ± 13 0.5  ± 0.1

Table III. Scatchard analysis of WT and mutant PTH-1 and PTH-2 receptors Homologous competition binding assays were performed in COS-7 cells for 6 h at 4 °C using 125I-[Nle8,21,Tyr34]rPTH-(1-34)amide as a tracer radioligand and varying amounts of the same unlabeled peptide as a competitor ligand. Values are means ± S.E. of the mean of four separate experiments. Receptor Binding affinity Kdapparent Surface PTH-(1-34)binding sites/cell nM ×106 P2R-WT 8.0  ± 1.7 0.5  ± 0.1 P2R-IL-244 3.5  ± 0.2 0.4  ± 0.1 P2Rc-YI-318 3.8  ± 0.8 1.5  ± 0.9 P1R-WT 10.1  ± 2.6 5.9  ± 2.0 P1R-LI-289 6.0  ± 0.7 3.0  ± 1.0 P1R-IY-363 11.3  ± 1.6 6.8  ± 1.5

To examine whether the ability to discriminate between His and Ile at position five could be conferred to the PTH-1 receptor, we introduced the reciprocal point mutations at the corresponding loci in the PTH-1 receptor and tested for effects on [Trp²³]PTHrP-(1-36) signaling. Neither mutation, Leu²⁸⁹  Ile (LI-289) nor Ile³⁶³  Tyr (IY-363), had any effect on His⁵ signaling selectivity, as the two mutant receptors exhibited full responsiveness to [Trp²³]PTHrP-(1-36) (Fig. 5, A-C). Interestingly, a position 5-specific effect of the IY-363 mutation was apparent in competition binding studies; the two PTHrP analogs displayed nearly equal potency in binding to the IY-363 mutant receptor (Fig. 5F and Table IV), whereas, [Trp²³]PTHrP-(1-36) was 2-5-fold weaker than [Ile⁵, Trp²³]PTHrP-(1-36) in binding to the other WT and mutant receptors (Figs. 4 and 5, and Table IV).

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Table IV. Ligand binding to WT and mutant PTH-1 and PTH-2 receptors Competition binding experiments were performed in COS-7 cells, using 125I-[Nle8,21,Tyr34]rPTH(1-34)amide as tracer and [Trp23,Tyr36] PTHrP(1-36)NH2 {[W23]PTHrP(1-36)} or [Ile5,Trp23,Tyr36]PTHrP(1-36)NH2 {[I5, W23]PTHrP(1-36)} as competitor peptides. Values are means (± S.E.) of the number of experiments indicated in parentheses. Receptor Competition binding IC50 [W23]PTHrP(1-36) [I5, W23]PTHrP(1-36) nM P2R-WT 39  ± 5  (13) 18  ± 3 (13) P2R-IL-244 30  ± 4  (13) 10  ± 1 (13) P2R-YI-318 65  ± 11  (9) 13  ± 2 (9) P1R-WT 100  ± 22  (8) 35  ± 7 (8) P1R-LI-289 125  ± 23  (7) 27  ± 5 (7) P1R-IY-363 29  ± 6  (7) 24  ± 5 (7)

Earlier studies with the PTH-1 receptor have indicated that PTH and PTHrP bind to overlapping sites (21-23). To examine whether the sites Ile²⁴⁴ and Tyr³¹⁸ in the PTH-2 receptor also have position 5-specific effects with PTH analogs, we studied the binding and signaling properties of [His⁵]PTH-(1-34) ([His⁵,Tyr³⁴]PTH-(1-34)amide) with the IL-244 and YI-318 mutant receptors. This PTH analog is inactive in cAMP assays with the WT PTH-2 receptor, although it binds with adequate affinity (IC₅₀ = 250 nM) (14). Both the IL-244 and YI-318 mutations caused at least a partial rescue of the signaling defect of [His⁵]PTH-(1-34) (Fig. 6, A-C). As was observed for the PTHrP analog, the corresponding mutations in the PTH-1 receptor had no effect on [His⁵]PTH-(1-34) signaling (Fig. 6, D-F), and the binding potencies that [His⁵]PTH-(1-34) displayed with the mutant PTH-2 or PTH-1 receptors were unaltered from the binding potencies seen with the corresponding WT receptor (data not shown).

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DISCUSSION

As part of our efforts to understand the mechanisms of ligand interaction in PTH receptors, we are exploring the molecular basis for the unique ligand selectivity of the PTH-2 receptor, which responds to PTH but not PTHrP. In the first stage of these studies, we examined the ligands for amino acid residue divergences that could explain this selectivity. We identified position 23 as the major determinant of binding selectivity, and position 5 as the major determinant of signaling selectivity (14). In the present study we sought to identify the sites in the PTH-2 receptor that enable it to discriminate, on the basis of cAMP signaling, between His and Ile at position 5. The receptor residues involved in this effect were of particular interest, because it seemed likely that they would be at, or near, sites involved in triggering the signal transduction mechanism.

The two PTHrP analogs used in our analysis differed by having His or Ile at position 5, and were thus nonfunctional or functional with the WT PTH-2 receptor. Each of the two analogs contained the Phe  Trp modification at position 23, which enabled high affinity binding of either analog to the PTH-2 receptor (14). The role of residue 5 in signaling selectivity was also demonstrated by Behar st al. (15). Importantly, this modification does not influence cAMP signaling, as demonstrated by the ability of [Trp²³,Tyr³⁶]PTHrP-(1-36)NH₂ (native histidine at position 5) to function as a PTH-2 receptor-selective antagonist (14). The Trp²³ modification thus permitted a direct analysis of the effects of residue 5 on signaling without the need to correct for weak binding affinities.

The cAMP responses of the two reciprocal PTH-1/PTH-2 receptor chimeras showed that the major determinants of His⁵/Ile⁵ signaling selectivity mapped to the portion of the receptor containing the membrane-spanning helices and connecting loops. Cassette mutagenesis then revealed three segments that affected this selectivity; these were located in extracellular loop 1, transmembrane helix 3, and extracellular loop 2. Scanning mutagenesis analysis of the extracellular loop 1 segment failed to reveal a single site that led to improved responsiveness to [Trp²³]PTHrP-(1-36), a finding that suggests that multiple residues in this region might be involved in residue 5 recognition. Point mutational analysis of the segments in transmembrane helix 3 and extracellular loop 2 successfully revealed two amino acids that strongly affected PTHrP signaling; Ile²⁴⁴ near the extracellular end of transmembrane helix 3 and Tyr³¹⁸ near the carboxyl-terminal end of extracellular loop 2 (Fig. 2). Changing either site in the PTH-2 receptor to the corresponding residue of the PTH-1 receptor resulted in a pronounced gain-of-function phenotype, as demonstrated by a selective increase in responsiveness to the [Trp²³]PTHrP-(1-36) analog.

The molecular basis by which Ile²⁴⁴ and Tyr³¹⁸ affect ligand selectivity is not known. The three-dimensional structure of the PTH-2 receptor, or any G protein-coupled receptor, with the exception of rhodopsin (24), has not been determined. The two-dimensional schematic of the core region of the PTH-2 receptor shown in Fig. 2 is based mainly on evolutionary and hydropathy analyses of the primary structure (25). Although the end points of the seven membrane-spanning helices have not been firmly established, it is predicted that Ile²⁴⁴ and Tyr³¹⁸ lie at, or close to, the boundary of the extracellular fluid and the lipid membrane. These two sites could thus be in a reasonable position for interacting with the ligand. Peptide-binding sites in another class of peptide hormone receptors, the tachykinin receptors, have been mapped to similar locations (13). Our functional data do not exclude the possibility that the mutations at Ile²⁴⁴ and Tyr³¹⁸ have allosteric effects on other residues, but global changes in receptor structure seem unlikely, because there was little or no effect of the mutations on the binding and signaling properties of [Ile⁵,Trp²³]PTHrP-(1-36) or rPTH-(1-34), or on receptor expression levels.

One question to consider in these studies is whether the PTH-2 receptor and the PTH-1 receptor engage their ligands in a similar fashion. In an effort to address this question we introduced reciprocal mutations, LI-289 and YI-363, into the PTH-1 receptor and tested the mutants for the ability to discriminate between analogs with His or Ile at position 5. No effect on cAMP signaling responsiveness was detected for either receptor mutation; we were thus unable to determine whether equivalent sites in the PTH-1 and PTH-2 receptors are involved in residue 5 recognition. That neither mutation was sufficient for conferring His⁵/Ile⁵ signaling selectivity to the PTH-1 receptor indicates that multiple PTH-2 receptor residues are required for this effect, as was suggested by the initial cassette mutagenesis studies in which mutations at three distinct sites led to a loss of selectivity (Fig. 3).

The bioactive regions of PTH and PTHrP differ considerably in primary structure (26), yet most studies indicate that the two ligands bind to the same site in the PTH-1 receptor (21-23). To examine whether the PTH-2 receptor sites that we identified here also influence interactions with position 5 of PTH, we studied the binding and signaling properties of [His⁵]hPTH-(1-34). This analog is inactive in cAMP assays with the PTH-2 receptor, though, it binds with adequate affinity (14). As with PTHrP, both the IL-244 and YI-318 mutations were able to rectify the signaling defect of [His⁵]PTH-(1-34) (Fig. 6). These results suggest that the histidine at position 5 in both [Trp²³]PTHrP-(1-36) and [His⁵]PTH-(1-34) is recognized by the same region of the PTH-2 receptor.

The isoleucine at position 244 is conserved in the rat and human PTH-2 receptor (1, 27), and leucine is preserved at the homologous site in each PTH-1 receptor (Xenopus, rat, mouse, human, porcine, and opossum). The same pattern of evolutionary preservation is seen for the Tyr³¹⁸ site in extracellular loop 2. Residue 5 in the ligands also shows this trend; the polar histidine is found here in all PTHrP ligands, and a hydrophobic isoleucine or methionine residue is found at the corresponding site in each vertebrate PTH sequence. It may be that the two receptor sites and the cognate residue 5 of the ligands are under the same evolutionary constraints. These constraints would ensure that PTHrP specifically interacts with the PTH-1 receptor to mediate its biological actions, including the regulation of embryonic bone development (28, 29) and not with the PTH-2 receptor, which is expressed in several different tissues, albeit with unknown functional consequences (27). Whether PTH is the actual ligand for the PTH-2 receptor is unknown; recent evidence suggests that the hypothalamus contains a novel peptide that selectively activates the PTH-2 receptor (30)

For the PTH receptors, and other members in this same peptide hormone receptor family, the amino-terminal extracellular domain has been shown to play an important role in ligand-binding affinity and specificity (3, 5, 8-10, 31, 32). Several other studies on these receptors have implicated the extracellular loops or transmembrane helices in ligand binding or signaling interactions (6-8, 10, 11). Our present studies with the PTH-2 receptor provide additional information on the functional map of the ligand interaction surface of the receptor, as they identify specific residues in helix 3 and extracellular loop 2 that modulate the signaling selectivity determined by residue 5 in the ligand.