The Hydrophobic Residues Phenylalanine 184 and Leucine 187 in the Type-1 Parathyroid Hormone (PTH) Receptor Functionally Interact with the Amino-terminal Portion of PTH-(1–34)*

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH2-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182–190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe184, Arg186, Leu187, and Ile190 as important determinants of maximum binding of 125I-labeled bovine PTH-(1–34) and125I-labeled bovine PTH-(3–34) and determinants of responsiveness to the NH2-terminal analog, PTH-(1–14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu22,Trp23]PTHrP-(15–36) to inhibit the cAMP response induced by PTH-(1–34). At Phe184 and Leu187, hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1–34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe184 and Leu187 in the PTH-1 receptor plays an important role in determining functional interaction with the 3–14 portion of PTH.

The PTH-1 1 receptor is a class II G protein-coupled receptor (1)(2)(3) that plays an important role in two distinct biological processes: the control of calcium ion concentrations in the blood and pattern formation in the developing skeleton (4). Most of the class II G protein-coupled receptors bind peptide hormones that are similar in size to the PTH-1 receptor agonists PTH-(1-34) and PTHrP- . Peptide ligands in this class include calcitonin-(1-32), secretin- , and glucagon- . The amino-terminal extracellular domains of the class II receptors are typically ϳ150 amino acids in length and contain several conserved amino acid sequence elements, including six cysteine residues. With all members of the class II G protein-coupled receptor family, mutagenesis studies have indicated that the NH 2 -terminal extracellular domain plays a dominant role in determining ligand binding affinity (5)(6)(7)(8)(9)(10), but the portions of these receptors containing the extracellular loops and transmembrane domains have also been shown to contribute to ligand binding (11)(12)(13)(14).
The receptor region at the COOH-terminal base of the extracellular domain (residues 182-190; cf. Fig. 1) was initially identified as a candidate ligand binding site by a homologscanning mutagenesis strategy. Replacement of this region of the rat PTH-1 receptor with the corresponding region of the secretin receptor abolished binding of PTH-(1-34) without affecting surface expression (23). Independent studies have demonstrated that a [Lys 13 (⑀-p-Bz 2 )]PTH-(1-34) analog crosslinked to this same region of the human PTH-1 receptor (24) and suggested that Arg 186 was the reactive site (25). In the current study, we explore further the role of nine amino acids in this juxtamembrane segment of the NH 2 -terminal domain of the rat PTH-1 receptor in determining the functional interaction with PTH-(1-34). The results reveal four receptor residues that modulate interaction with the 3-14 portion of PTH and suggest that hydrophobicity is required for optimal ligandbinding and cAMP-signaling potency at Phe 184 and Leu 187 .  ) were prepared on an Applied Biosystems model 431A peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) protecting group chemistry and trifluoroacetic acid-mediated cleavage/deprotection (MGH Biopolymer Synthesis Facility, Boston, MA); these peptide were then purified by high performance liquid chromatography and lyophilized. The peptide rPTH-(1-14)NH 2 (PTH- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)) was synthesized on a multiple peptide synthesizer (Advanced Chemtech model 396 MBS) and desalted by adsorption on a C18containing cartridge (Sep-Pak). All peptides were reconstituted in 10 mM acetic acid and stored at ϳ80°C. The purity, identity, and stock concentration of each compound was secured by analytical high per-formance liquid chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and amino acid analysis. Radiolabeling of PTH-(1-34) and PTH-(3-34) was performed using 125 I-Na (2200 Ci/ mmol; NEN Life Science Products) and chloramine-T; the resultant 125 I-labeled ligand was purified by high performance liquid chromatography.

Peptides
PTH Receptor Mutagenesis-The construction and initial characterization of the pCDNA-1-based (InVitrogen, San Diego, CA) plasmids encoding the intact epitope-tagged rat PTH-1 receptor (rWT-HA, or wild type) has been described previously (5). The HA tag in rWT-HA is a nine-amino acid sequence that replaces residues 93-101 in the receptor's extracellular domain and does not affect receptor function (5). Point mutations were incorporated into single-strand wild type receptor plasmid DNA by oligonucleotide-directed mutagenesis (26). The nucleotide sequence of each mutant plasmid was verified by the dideoxynucleotide chain termination method using single-stranded plasmid DNA as template.
The construction and initial characterization of the pCDNA-1-based plasmid encoding the epitope-tagged amino-terminally truncated rat PTH-1 receptor (r⌬Nt-HA) has also been described previously (22). In this receptor, residues 23-181 have been removed, and a nine-amino acid HA tag joined to a tetraglycine linker (YPYDVPDYAGGGG-) has been inserted between Ala 22 and Glu 182 . Signal peptidase cleavage is predicted to occur between Ala 22 and the tyrosine of the HA tag (27). The Phe 184 point mutation was incorporated into r⌬Nt-HA as described above.
Cell Culture and DNA Transfection-Stock solutions of EGTA/trypsin and antibiotics were from Life Technologies, Inc.; fetal bovine serum was from Hyclone Laboratories (Logan, UT). COS-7 cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10%); penicillin G (20 units/ml), streptomycin sulfate (20 g/ml), and amphotericin B (0.05 g/ml) in a humidified atmosphere containing 5% CO 2 . Transient transfections of COS-7 cells were performed using DEAE-dextran as described previously (17). 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. Twenty-four to sixteen hours prior to assay, the cells were treated with fresh media and shifted to a humidified incubator containing 5% CO 2 that was set at 33°C (17,28). Assays were conducted 72-96 h after transfection, at which point ϳ20% of the cells were transfected and expressed surface wild type PTH receptors at a density of about 5 ϫ 10 6 /cell (17).
Ligand-binding Assays-Binding reactions were performed with transiently transfected COS-7 cells in 24-well plates. 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 successively with 200 l of binding buffer and 100 l of binding buffer containing ϳ100,000 cpm of 125 I-tracer (ϳ26 fmol; final volume ϭ 300 l). The cells were incubated at 15°C for 4 h and placed on ice; the binding medium was removed, and the monolayer was rinsed three times with 0.5 ml of cold binding buffer and lysed with 0.5 ml of 5 N NaOH. The entire lysate was counted for ␥-irradiation. Nonspecific binding was determined in cells transfected with the pCDNA-1 vector and was typically less than 1.5% of the total radioactivity added.
PTH-1 Receptor Expression-Measurements of surface expression for the HA epitope-tagged receptors by indirect antibody binding methods was performed with intact transfected COS-7 cells in 24-well plates. Cells were washed with 0.5 ml of binding buffer and then incubated with 0.25 ml of binding buffer containing the mouse monoclonal antibody 12CA5 (Roche Molecular Biochemicals) at 1 g/ml for 2 h at 15°C. The buffer was removed, and the cells were then washed three times with 0.5 ml of binding buffer and incubated for an additional 2 h at 15°C with 0.25 ml of binding buffer containing ϳ400,000 cpm of 125 Ilabeled goat anti-mouse IgG antibody (NEN Life Science Products). The buffer was withdrawn, and cells were then washed three times with 0.5 ml of binding buffer and lysed with 5 N NaOH. The entire lysate was counted for ␥-irradiation. Nonspecific binding was determined in cells transfected with the pCDNA-1 vector and was typically less than 0.5% of the total radioactivity added.
Intracellular Cyclic AMP-Stimulation of transiently transfected COS-7 cells was performed in 24-well plates. Cells were rinsed with 0.5 ml of binding buffer and treated with 200 l of cAMP assay buffer (Dulbecco's modified Eagle's medium containing 2 mM 3-isobutyl-1methylxanthine, 1 mg/ml bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4), and 100 l of binding buffer containing varying amounts of peptide analog (final volume ϭ 300 l). The medium was removed after incubation for 30 min at 37°C, and the cells were frozen (Ϫ80°C), lysed with 0.5 ml of 50 mM HCl, and refrozen (Ϫ80°C). The cAMP content of the diluted lysate was determined by radioimmunoassay using unlabeled cAMP as a standard. The cAMP EC 50 values were determined using nonlinear regression (see below).
Inhibition Studies-The cAMP stimulation protocol described above was utilized for inhibition studies with some minor modifications. Cells were rinsed with 0.5 ml of binding buffer and treated successively with 100 l of cAMP assay buffer, 50 l of binding buffer containing varying doses of [Glu 22 ,Trp 23 ]PTHrP , and 100 l of cAMP assay buffer containing a 1 nM dose of rPTH-(1-34) (final volume ϭ 250 l). Cells were incubated for 30 min at room temperature and processed as above. The dose of [Glu 22 ,Trp 23 ]PTHrP-(15-36) that inhibited the PTH-(1-34)mediated cAMP response by 50% (IC 50A ) was calculated using nonlinear regression analysis (see below).
Data Calculation-All calculations were performed using Microsoft ® Excel. Nonlinear regression analysis of binding and cAMP stimulation data was performed using four parameters, defined as the Minimum (Min), Maximum (Max), midpoint (IC 50 ), and slope of the response curve. The predicted response (y p ) for a given dose (x) of peptide was calculated using the following equation: y p ϭ Min ϩ ((Max Ϫ Min)/(1 ϩ (IC 50 /x) slope )). The initial parameter values were estimated from the primary data, and the Excel "Solver function" was then used to vary the four parameters in order to minimize the differences between the predicted and actual responses (least-squares method) (29). 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
We introduced individual alanine substitutions at each position in the 182-190 region of the rat PTH-1 receptor (Fig. 1) and analyzed the effects on receptor function in transiently transfected COS-7 cells (Table I). The surface expression of the alanine-substituted mutants ranged from 85 to 113% of the wild type receptor, as judged by antibody-binding analysis (Table I). Four of the mutations (Phe 184 3 Ala, Arg 186 3 Ala, Leu 187 3 Ala, and Ile 190 3 Ala) reduced the capacity of the receptor to bind the agonist tracer 125 I-PTH-(1-34) by 4-fold or more (Fig. 2). The strongest effect occurred with the Phe 184 3 Ala mutation, which reduced binding to 4 Ϯ 0.4% of the binding seen with the wild type receptor. A similar pattern was observed when the alanine-substituted mutant receptors were tested for their capacity to bind the partial agonist tracer 125 I-PTH-(3-34) ( Table I). Each of the nine alanine-substituted mutant receptors mediated a comparable maximal (40-fold) increase in intracellular cAMP in response to high doses of PTH-(1-34), as was observed with the wild type receptor ( Table  I). The cAMP-stimulating potency of PTH (1-34) with most of the alanine-substituted mutants was similar to the potency seen with the wild type receptor (EC 50 1.4 Ϯ 0.3 nM, Table I), but the Phe 184 3 Ala mutation resulted in an 8-fold decrease in potency of PTH-(1-34) agonist peptide relative to rWT-HA (p ϭ 0.02, Fig. 3).
In order to analyze the effect of the alanine mutations on the NH 2 -terminal signaling domain of PTH-(1-34), we utilized the COOH-terminally truncated rPTH-(1-14)NH 2 . As reported previously (22), stimulation of rWT-HA with a 100 M dose of PTH-(1-14) induced a 14-fold increase in cAMP formation relative to the basal response. Stimulation of the alanine-substituted mutants with the same dose of PTH-(1-14) revealed that Phe 184 3 Ala, Arg 186 3 Ala, Leu 187 3 Ala, and Ile 190 3 Ala each showed a 7-20-fold reduced responsiveness to this peptide in cAMP assays relative to rWT-HA (p Ͻ 0.0001, Fig. 4).
To   To determine if other residues in the NH 2 -terminal extracellular domain of the PTH-1 receptor were required for the functional effects observed for the mutations in the 182-190 region, we utilized a truncated rat PTH-1 receptor (r⌬Nt-HA) that lacked residues 23-181 and had in their place a nine-amino acid HA epitope tag (Fig. 6A) (22). Introduction of the Phe 184 3 Ala mutation into r⌬Nt-HA yielded a truncated mutant receptor that was expressed on the cellular surface to the same level as unsubstituted r⌬NT-HA (Fig. 6B). The unsubstituted truncated receptor elicited ϳ6-fold increases in cAMP levels in response to either 1 M PTH-(1-34) or 100 M PTH-(1-14); r⌬Nt-HA(FA-184) exhibited little or no response to these peptides (Fig. 6C).
In order to characterize the chemical basis for the role of the 182-190 region in interacting with PTH-(1-34), we examined the effects of a number of polar and nonpolar mutations in the 184 -187 segment of intact rWT-HA (Table I and Fig. 7). All of these point mutations yielded mutant receptors that were well expressed on the cell surface (range ϭ 78 -120% of rWT-HA, Table I

DISCUSSION
The present study was conducted to explore the functional role(s) of individual residues in the (182-190) juxtamembrane region of the NH 2 -terminal domain of the PTH-1 receptor. The potential importance of this segment of the receptor was recognized previously in a homolog-scanning mutagenesis study (23) and has also been investigated by photochemical crosslinking (24,25) and spectroscopic methods (31). In the current study, we extended the functional analysis by first performing an alanine-scanning experiment, the results of which suggested that Phe 184 , Arg 186 , Leu 187 , and Ile 190 played a role in the optimal binding of 125 I-PTH-(1-34) (Fig. 2), 125 I-PTH-(3-34) (Table I), and 125 I-PTHrP-(1-36) (data not shown). Additional point mutations targeted to this region verified the importance of Phe 184 and Leu 187 and suggested that side chain hydrophobicity at these positions is a key determinant of ligand/receptor interaction (Table I).  (1-14). The wild type and alanine-substituted mutant PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with a 100 M dose of rPTH- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Shown are data (mean Ϯ S.E.) combined from three individual experiments, each of which was performed in duplicate on a separate day, as described under "Materials and Methods." The basal (unstimulated) level of cAMP in these cells ranged from 10 Ϯ 1 pmol/well (LA-187) to 15 Ϯ 2 pmol/well (MA-189) and was not subtracted in the calculation. Ⅺ, untreated; f, treated with 100 M rPTH-(1-14).
All of the mutant receptors in this study were expressed near wild type levels on the surface of transfected COS-7 cells, as judged by antibody binding. The decreases in maximum specific binding of radiolabeled PTH tracers observed for several of these mutants suggest that certain substitutions in the 182-190 region reduce ligand binding affinity. The low specific binding of radiolabeled PTH-(1-34) (less than 3% of total counts added) observed with the key mutants prevented us from performing meaningful competition binding assays. However, Phe 184 3 Ala, Arg 186 3 Ala, Leu 187 3 Ala, and Ile 190 3 Ala did allow for competitive antagonism of the PTH-(1-34)induced cAMP response by the weak-binding fragment [Glu 22 ,Trp 23 ]PTHrP-(15-36) (Fig. 5); this is consistent with the weakened binding of PTH-(1-34). In addition, for the mutations that reduced the specific binding of 125 I-bPTH-(1-34) to less than 6% of that seen with the wild type receptor, the cAMP-stimulating potencies of PTH-(1-34) were 8 -300-fold weaker than that seen for the wild type receptor (Table I).
These results are consistent with the above mutant receptors exhibiting a reduced affinity for PTH-(1-34), although the possibility of additional activation-specific effects caused by the mutations cannot be excluded.
Recently, Pellegrini, et al. (31) determined the structure of a synthetic peptide containing residues 168 -198 of the PTH-1 receptor in a micellar solution of dodecylphosphocholine using NMR spectroscopy and demonstrated that residues 180 -189 of this receptor fragment formed an amphipathic ␣-helix, which was lipid-associated. These authors suggested that the solventexposed hydrophilic face of this helix interacts with charged residues in the COOH-terminal portion of PTH-(1-34) (31). Our current functional data are most consistent with the view that residues Phe 184 , Arg 186 , Leu 187 , and Ile 190 in the PTH-1 receptor interact with the 3-14 region of PTH-(1-34) and that this interaction is important for ligand binding and ligandinduced cAMP signaling. If the 182-190 region of the intact receptor were to be ␣-helical, than these four critical residues at positions 184, 186, 187, and 190 would form a contiguous surface; the results of our substitution analysis would suggest that the hydrophobicity of Phe 184 and Leu 187 and possibly the aliphatic portion of the side chain of Arg 186 contribute to the functionality of this surface. Our mutational data do not allow us to distinguish whether or not these four residues exert their effects on ligand function through a direct or an indirect mechanism, but the cross-linking of a [Lys 13 (⑀-p-Bz 2 )]PTH-(1-34) analog to Arg 186 in the PTH-1 receptor (25) suggests that some contact could occur between this receptor surface and the ligand. Further work is clearly needed to resolve how the residues in this domain of the PTH-1 receptor contribute to the structure of the receptor and to its interaction with PTH ligands and to determine whether the corresponding regions of other class II receptors are involved in analogous interactions with their respective peptide ligands.