HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carter, P. H. Articles by Gardella, T. J. Search for Related Content PubMed PubMed Citation Articles by Carter, P. H. Articles by Gardella, T. J. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 45, 31955-31960, November 5, 1999

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)^*

Percy H. Carter, Masaru Shimizu, Michael D. Luck, and Thomas J. Gardella

From the Endocrine Unit, Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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) and ¹²⁵I-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The PTH-1¹ receptor is a class II G protein-coupled receptor (1-3) 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-(1-36). Peptide ligands in this class include calcitonin-(1-32), secretin-(1-27), and glucagon-(1-29). The amino-terminal extracellular domains of the class II receptors are typically ~150 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 NH₂-terminal extracellular domain plays a dominant role in determining ligand binding affinity (5-10), but the portions of these receptors containing the extracellular loops and transmembrane domains have also been shown to contribute to ligand binding (11-14).

Several groups are utilizing a variety of approaches to identify specific points of contact between PTH-(1-34) and the PTH-1 receptor. Both mutational and photochemical cross-linking studies on the PTH-1 receptor have suggested that portions of the NH₂-terminal extracellular receptor domain interact with the COOH-terminal (positions 15-34) binding domain of PTH (8, 15); similar observations have been made for the other class II receptors (16, 17). Separate mutational and cross-linking studies have indicated that portions of the extracellular loops of the PTH-1 receptor contact residues within the amino-terminal (positions 1-14) portion of the ligand (17-21) and that this interaction is sufficient for stimulating the cellular cAMP response (22).

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 homolog-scanning 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 [Lys¹³(-p-Bz₂)]PTH-(1-34) analog cross-linked to this same region of the human PTH-1 receptor (24) and suggested that Arg¹⁸⁶ was the reactive site (25). In the current study, we explore further the role of nine amino acids in this juxtamembrane segment of the NH₂-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 ligand-binding and cAMP-signaling potency at Phe¹⁸⁴ and Leu¹⁸⁷.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Peptides-- [Nle^8,18,Tyr³⁴]bPTH-(3-34)NH₂ (PTH-(3-34)) was purchased from Bachem (Torrance, CA). [Nle^8,18,Tyr³⁴]bPTH-(1-34)NH₂ (PTH-(1-34)), and [Glu²²,Trp²³,Tyr³⁶]human PTHrP-(15-36)NH₂ ([Glu²²,Trp²³]PTHrP-(15-36)) 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)NH₂ (PTH-(1-14)) was synthesized on a multiple peptide synthesizer (Advanced Chemtech model 396 MBS) and desalted by adsorption on a C18-containing cartridge (Sep-Pak). All peptides were reconstituted in 10 mM acetic acid and stored at ~80 °C. The purity, identity, and stock concentration of each compound was secured by analytical high performance 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 ¹²⁵I-Na (2200 Ci/mmol; NEN Life Science Products) and chloramine-T; the resultant ¹²⁵I-labeled ligand was purified by high performance liquid chromatography.

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 (rNt-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 Ala²² and Glu¹⁸². Signal peptidase cleavage is predicted to occur between Ala²² and the tyrosine of the HA tag (27). The Phe¹⁸⁴ point mutation was incorporated into rNt-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 µg/ml), and amphotericin B (0.05 µg/ml) in a humidified atmosphere containing 5% CO₂. 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% CO₂ that was set at 33 °C (17, 28). Assays were conducted 72-96 h after transfection, at which point ~20% of the cells were transfected and expressed surface wild type PTH receptors at a density of about 5 × 10⁶/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 CaCl₂, 5% heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH 7.7 with HCl) and treated successively with 200 µl of binding buffer and 100 µl of binding buffer containing ~100,000 cpm of ¹²⁵I-tracer (~26 fmol; final volume = 300 µl). 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 µg/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 ~400,000 cpm of ¹²⁵I-labeled 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 µl of cAMP assay 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 containing varying amounts of peptide analog (final volume = 300 µl). The medium was removed after incubation for 30 min at 37 °C, and the cells were frozen (80 °C), lysed with 0.5 ml of 50 mM HCl, and refrozen (80 °C). The cAMP content of the diluted lysate was determined by radioimmunoassay using unlabeled cAMP as a standard. The cAMP EC₅₀ 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 µl of cAMP assay buffer, 50 µl of binding buffer containing varying doses of [Glu²²,Trp²³]PTHrP (15-36), and 100 µl of cAMP assay buffer containing a 1 nM dose of rPTH-(1-34) (final volume = 250 µl). Cells were incubated for 30 min at room temperature and processed as above. The dose of [Glu²²,Trp²³]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 (IC₅₀), 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 = Min + ((Max  Min)/(1 + (IC₅₀/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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (Phe¹⁸⁴  Ala, Arg¹⁸⁶  Ala, Leu¹⁸⁷  Ala, and Ile¹⁹⁰  Ala) reduced the capacity of the receptor to bind the agonist tracer ¹²⁵I-PTH-(1-34) by 4-fold or more (Fig. 2). The strongest effect occurred with the Phe¹⁸⁴  Ala mutation, which reduced binding to 4 ± 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 ¹²⁵I-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 (EC₅₀ 1.4 ± 0.3 nM, Table I), but the Phe¹⁸⁴  Ala mutation resulted in an 8-fold decrease in potency of PTH-(1-34) agonist peptide relative to rWT-HA (p = 0.02, Fig. 3).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Schematic of the PTH-1 receptor. Shown is the predicted domain structure of the rat PTH-1 receptor used in this study and the locations of the eight conserved extracellular cysteines (C) and the nine amino acid HA epitope tag (hatched circles). The expanded view shows the nine-amino acid segment at the juxtamembrane end of the amino-terminal extracellular domain of the receptor (residues 182-190), which was subjected to point mutational analysis, as described under "Materials and Methods" and in succeeding figures. The boundary of the NH2-terminal domain and the first transmembrane domain (TM-1) is shown as predicted by the Proteinpredict algorithm (32).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of alanine mutations in the PTH-1 receptor on ligand binding. The wild type and mutant rat PTH-1 receptors bearing alanine point substitutions at positions 182-190 were transiently transfected into COS-7 cells and functionally evaluated. The maximum specific binding of 125I-bPTH-(1-34) to these receptors is shown and is expressed as the percentage of the specific binding observed for that ligand with the wild type receptor. Shown are data (mean ± S.E.) combined from three individual experiments, each of which was performed in duplicate on a separate day, as described under "Materials and Methods." Nonspecific binding was subtracted in the calculation.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Dose-response analysis of PTH-(1-34) stimulation of wild type and mutant PTH-1 receptors. Wild type or alanine-substituted mutant PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with bPTH-(1-34) over the dose range indicated. Shown are data (mean ± 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 combined rWT-HA curve (n = 3, dashed line) matched for this particular experiment is shown as a control. , rWT-HA; , FA-184; , RA-186; , LA-187; , IA-190.

In order to analyze the effect of the alanine mutations on the NH₂-terminal signaling domain of PTH-(1-34), we utilized the COOH-terminally truncated rPTH-(1-14)NH₂. As reported previously (22), stimulation of rWT-HA with a 100 µM 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 Phe¹⁸⁴  Ala, Arg¹⁸⁶  Ala, Leu¹⁸⁷  Ala, and Ile¹⁹⁰  Ala each showed a 7-20-fold reduced responsiveness to this peptide in cAMP assays relative to rWT-HA (p < 0.0001, Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of alanine mutations in the PTH-1 receptor on stimulation by PTH-(1-14). The wild type and alanine-substituted mutant PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with a 100 µM dose of rPTH-(1-14). Shown are data (mean ± 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 ± 1 pmol/well (LA-187) to 15 ± 2 pmol/well (MA-189) and was not subtracted in the calculation. , untreated; , treated with 100 µM rPTH-(1-14).

To analyze the effect of the alanine mutations on interactions involving the COOH-terminal binding domain of PTH-(1-34), we utilized the fragment [Glu²²,Trp²³,Tyr³⁶]PTHrP-(15-36)NH₂ (30) and tested for its ability to block the cAMP response induced by a 1 nM dose of PTH-(1-34) with each mutant receptor. With rWT-HA, a 10 µM dose of [Glu²²,Trp²³,Tyr³⁶] PTHrP-(15-36)NH₂ did not significantly inhibit the cAMP response induced by PTH-(1-34) (Fig. 5A), a result that is consistent with the weak ability of this fragment to inhibit the binding of ¹²⁵I-PTH-(1-34) to COS-7 cells transfected with rWT-HA (IC₅₀ >1000 nM) (30). With the mutants containing alanine-substitutions at positions 184, 186, 187, and 190, a 10 µM dose of [Glu²²,Trp²³Tyr³⁶]PTHrP-(15-36)NH₂ significantly inhibited the PTH-(1-34)-mediated response (Fig. 5A). This inhibition was dose-dependent and attained 50% reduction of the control response at doses of 2.9 ± 0.3, 3.8 ± 1.1, and 11.0 ± 2.9 µM, for the Phe¹⁸⁴  Ala, Leu¹⁸⁷  Ala, and Ile¹⁹⁰  Ala mutants, respectively (Fig. 5B). When a 10 µM dose of [Glu²²,Trp²³]PTHrP-(15-36) was tested against a full-range dose response curve of PTH-(1-34) with rWT-HA, Phe¹⁸⁴  Ala and Ile¹⁹⁰  Ala, a rightward shift in cAMP-stimulating potency was observed only for the alanine-substituted mutants (data not shown); since this rightward shift was not accompanied by a reduction in cAMP-stimulating efficacy of PTH-(1-34), the inhibition of PTH-(1-34) by [Glu²²,Trp²³]PTHrP-(15-36) is most likely occurring through a competitive binding mechanism.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Antagonism of the PTH-induced cAMP response by [Glu22,Trp23]PTHrP-(15-36) with mutant PTH-1 receptors. A, wild type and alanine-substituted mutant PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with a 1 nM dose of bPTH-(1-34) in the presence () or absence () of a 10 µM dose of [Glu22,Trp23]PTHrP-(15-36). Shown are data (mean ± 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 ± 0.5 (FA-184 receptor) to 14 ± 0.7 pmol/well (IA-190 receptor) and was not subtracted in the calculation. Differences between the antagonized and unantagonized responses that are demarcated with an asterisk are significant (p < 0.0005), while all others are not (p > 0.15). B, the wild type (), FA-184 (), LA-187 (), and IA-190 () PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with a 1 nM dose of bPTH-(1-34) in the presence of varying doses of [Glu22,Trp23]PTHrP-(15-36). Shown are data (mean ± 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 8.3 ± 1.2 (FA-184 receptor) to 9.7 ± 1.5 pmol/well (rWT-HA) and was not subtracted in the calculation.

To determine if other residues in the NH₂-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 (rNt-HA) that lacked residues 23-181 and had in their place a nine-amino acid HA epitope tag (Fig. 6A) (22). Introduction of the Phe¹⁸⁴  Ala mutation into rNt-HA yielded a truncated mutant receptor that was expressed on the cellular surface to the same level as unsubstituted rNT-HA (Fig. 6B). The unsubstituted truncated receptor elicited ~6-fold increases in cAMP levels in response to either 1 µM PTH-(1-34) or 100 µM PTH-(1-14); rNt-HA(FA-184) exhibited little or no response to these peptides (Fig. 6C).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of the FA-184 mutation in a PTH-1 receptor lacking the majority of the extracellular domain. A, a schematic diagram of the truncated receptor. The amino-terminal nine-amino acid HA tag (hatched) is joined by a Gly4 spacer to Glu182 of the receptor. B, the specific binding of antibody 12CA5 and a radiolabeled second antibody to the transiently expressed rWT-HA full-length control and the two r(Nt)-HA epitope-tagged receptors is illustrated. Shown are data (mean ± S.E.) combined from three individual experiments, each of which was performed in triplicate on a separate day, as described under "Materials and Methods." Nonspecific binding was not subtracted in the calculation. C, two receptors were transfected into COS-7 cells and subsequently stimulated with either a 1 µM dose of bPTH-(1-34) or a 100 µM dose of rPTH-(1-14). Shown are data (mean ± S.E.) combined from two individual experiments, each of which was performed in triplicate on a separate day, as described under "Materials and Methods." The basal (unstimulated) level of cAMP in these cells was not subtracted in the calculation and is also illustrated. Hatched bar, untreated; black bar, 1 µM PTH-(1-34); gray bar, 100 µM PTH-(1-14).

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 = 78-120% of rWT-HA, Table I). Substitution of the polar amino acids Glu, Lys, and Arg at position 184 resulted in 10-50-fold reductions in the maximal specific binding of ¹²⁵I-PTH-(1-34), the cAMP-signaling potency of PTH-(1-34), and the cAMP-signaling efficacy of PTH-(1-14) (Table I and Fig. 7). Substitution of the nonpolar amino acids Ile, Met, and Leu at position 184 resulted in less severe (~2-fold) reductions in PTH-(1-34) binding and PTH-(1-14) stimulation and did not significantly impact the stimulation by PTH-(1-34). Mutation of Asp¹⁸⁵ to either Ile or Lys did not alter the binding or signaling properties of PTH analogs. In accord with the findings of Adams and co-workers (25), the introduction of lysine at position 186 was well tolerated in both binding and signaling assays. The Arg¹⁸⁶  Ile mutant showed a 2-fold reduction in the maximum specific binding of ¹²⁵I-PTH-(1-34) and a 7.5-fold reduction in the specific binding of ¹²⁵I-PTH (3-34), as compared with rWT-HA, but responded normally to PTH-(1-34) in cAMP stimulation assays. Interestingly, the Arg¹⁸⁶  Ile mutant receptor was less functionally impaired than the Arg¹⁸⁶  Ala receptor. Substitution of the polar residue Glu, His, or Arg at position 187 with Ala resulted in severe reductions (>15-fold) in PTH-(1-34) binding capacity and the cAMP-stimulation potency of PTH-(1-34), whereas introduction of Ile and Val at this position resulted in small (~2-fold) or no reductions in PTH-(1-34)-binding capacity or cAMP signaling potency of PTH-(1-34).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Dose-response analysis of PTH-(1-34) stimulation of mutant PTH-1 receptors. Wild type or mutant PTH-1 receptors were transfected into COS-7 cells and subsequently stimulated with bPTH-(1-34) over the dose range indicated. Shown are data (mean ± 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 combined rWT-HA curve (n = 3, dashed line) matched for each particular set of experiments is shown in each panel as a control. A, , rWT-HA; , FI-184; , FK-184; , FE-184. B, , rWT-HA; , DI-185; , DK-185. C, , rWT-HA; , RI-186; , RK-186. D, , rWT-HA; , LE-187; , LI-187; , LR-187.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study was conducted to explore the functional role(s) of individual residues in the (182-190) juxtamembrane region of the NH₂-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 cross-linking (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 Phe¹⁸⁴, Arg¹⁸⁶, Leu¹⁸⁷, and Ile¹⁹⁰ played a role in the optimal binding of ¹²⁵I-PTH-(1-34) (Fig. 2), ¹²⁵I-PTH-(3-34) (Table I), and ¹²⁵I-PTHrP-(1-36) (data not shown). Additional point mutations targeted to this region verified the importance of Phe¹⁸⁴ and Leu¹⁸⁷ and suggested that side chain hydrophobicity at these positions is a key determinant of ligand/receptor interaction (Table I).

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, Phe¹⁸⁴  Ala, Arg¹⁸⁶  Ala, Leu¹⁸⁷  Ala, and Ile¹⁹⁰  Ala did allow for competitive antagonism of the PTH-(1-34)-induced cAMP response by the weak-binding fragment [Glu²²,Trp²³]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 ¹²⁵I-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.

To localize the region of the ligand affected by the mutations in the 182-190 region of the receptor, we examined the ability of the mutant receptors to interact with PTH ligands of varying length. The Phe¹⁸⁴  Ala, Arg¹⁸⁶  Ala, Leu¹⁸⁷  Ala, and Ile¹⁹⁰  Ala receptors each (i) reduced the specific binding of ¹²⁵I-PTH-(3-34), (ii) reduced the cAMP-signaling responsiveness to PTH-(1-14), and (iii) enhanced the ability of [Glu²²,Trp²³,Tyr³⁶]PTHrP-(15-36)NH₂ to inhibit the cAMP response mediated by PTH-(1-34). In addition, every receptor in this study that exhibited impaired capacity to bind ¹²⁵I-PTH-(3-34) also demonstrated reduced responsiveness to PTH-(1-14) (Table I). These data suggest that the mutations in the 182-190 region of the PTH-1 receptor alter interactions with the 3-14 portion of PTH-(1-34). These receptor mutations at the COOH-terminal end of the amino-terminal domain thus stand in contrast to previously described PTH-1 receptor mutations at the receptor's extreme amino terminus (e.g. Thr³³  Ala, which impaired the binding of PTH-(1-34) but did not affect PTH-(1-14) signaling), and in the third extracellular loop (e.g. Trp⁴³⁷  Ala, which impaired the binding of PTH-(1-34) but not PTH-(3-34)) (15, 22, 23).

Deletion of most of the amino-terminal domain of the PTH-1 receptor (residues 23-181) abolishes detectable binding of ¹²⁵I-labeled PTH analogs; however, we recently showed that surface expression levels and cAMP-signaling responsiveness to PTH-(1-34) and PTH-(1-14) are partially preserved in this truncated r(Nt)-HA receptor (22). The insertion of the Phe¹⁸⁴  Ala mutation into r(Nt)-HA severely diminished the cAMP-signaling responses to both PTH-(1-34) and PTH-(1-14) without affecting receptor expression. These data indicate that the role of Phe¹⁸⁴ in r(Nt)-HA is homologous with its role in the full-length PTH-1 receptors and thus suggest that Phe¹⁸⁴ does not depend on the major portion of the amino-terminal domain for its interaction with PTH-(1-34).

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 solvent-exposed 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 Phe¹⁸⁴, Arg¹⁸⁶, Leu¹⁸⁷, and Ile¹⁹⁰ 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 ligand-induced 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 Phe¹⁸⁴ and Leu¹⁸⁷ and possibly the aliphatic portion of the side chain of Arg¹⁸⁶ 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 [Lys¹³(-p-Bz₂)]PTH-(1-34) analog to Arg¹⁸⁶ 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.

	ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. John T. Potts, Jr. and Ernestina Schipani for reading the manuscript and providing insightful discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK11794.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-726-3683; E-mail: Gardella@helix.MGH.Harvard.edu; Fax: 617-726-7543.

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; rPTH, rat PTH; bPTH, bovine PTH; PTHrP, PTH-related peptide; Nle, norleucine; Bz, benzoyl; WT, wild type; rWT, rat wild type; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. J. Fitzsimmons, X. Zhao, and S. A. Wank The Extracellular Domain of Receptor Activity-modifying Protein 1 Is Sufficient for Calcitonin Receptor-like Receptor Function J. Biol. Chem., April 11, 2003; 278(16): 14313 - 14320. [Abstract] [Full Text] [PDF]

				J.-P. Vilardaga, I. Lin, and R. A. Nissenson Analysis of Parathyroid Hormone (PTH)/Secretin Receptor Chimeras Differentiates the Role of Functional Domains in the PTH/ PTH-Related Peptide (PTHrP) Receptor on Hormone Binding and Receptor Activation Mol. Endocrinol., July 1, 2001; 15(7): 1186 - 1199. [Abstract] [Full Text] [PDF]

				M. Shimizu, P. H. Carter, A. Khatri, J. T. Potts Jr., and T. J. Gardella Enhanced Activity in Parathyroid Hormone-(1-14) and -(1-11): Novel Peptides for Probing Ligand-Receptor Interactions Endocrinology, July 1, 2001; 142(7): 3068 - 3074. [Abstract] [Full Text] [PDF]

				P. H. Carter, B. D. Petroni, R. C. Gensure, E. Schipani, J. T. Potts Jr., and T. J. Gardella Selective and Nonselective Inverse Agonists for Constitutively Active Type-1 Parathyroid Hormone Receptors: Evidence for Altered Receptor Conformations Endocrinology, April 1, 2001; 142(4): 1534 - 1545. [Abstract] [Full Text]

				M. Shimizu, P. H. Carter, and T. J. Gardella Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand J. Biol. Chem., June 23, 2000; 275(26): 19456 - 19460. [Abstract] [Full Text] [PDF]

				N. Shimizu, J. Guo, and T. J. Gardella Parathyroid Hormone (PTH)-(1-14) and -(1-11) Analogs Conformationally Constrained by alpha -Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor J. Biol. Chem., December 21, 2001; 276(52): 49003 - 49012. [Abstract] [Full Text] [PDF]

				M. Shimizu, J. T. Potts Jr., and T. J. Gardella Minimization of Parathyroid Hormone. NOVEL AMINO-TERMINAL PARATHYROID HORMONE FRAGMENTS WITH ENHANCED POTENCY IN ACTIVATING THE TYPE-1 PARATHYROID HORMONE RECEPTOR J. Biol. Chem., July 14, 2000; 275(29): 21836 - 21843. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carter, P. H. Articles by Gardella, T. J. Search for Related Content PubMed PubMed Citation Articles by Carter, P. H.