©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Truncation of the Carboxyl-terminal Region of the Rat Parathyroid Hormone (PTH)/PTH-related Peptide Receptor Enhances PTH Stimulation of Adenylyl Cyclase but Not Phospholipase C (*)

(Received for publication, October 27, 1994; and in revised form, February 6, 1995)

Akiko Iida-Klein Jun Guo Lin Y. Xie Harald Jüppner John T. Potts Jr. Henry M. Kronenberg F. Richard Bringhurst Abdul B. Abou-Samra Gino V. Segre (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent K values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 ± 0.19 nM; R513, 1.24 ± 0.12 nM; R480, 0.48 ± 0.05 nM, mean ± S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with G(s).


INTRODUCTION

The parathyroid hormone (PTH)(^1)/PTH-related peptide (PTHrP) receptor (1, 2) belongs to an unique family within the seven membrane-spanning guanine-nucleotide regulatory protein (G protein)-coupled receptor superfamily. This family includes mammalian receptors for calcitonin (3) , secretin(4) , glucagon(5) , glucagon-like peptide-1(6) , growth hormone-releasing hormone(7) , vasoactive intestinal peptide(8) , vasoactive intestinal peptide-2(9) , pituitary adenylyl cyclase-activating peptide(10) , gastric inhibitory peptide(11) , and corticotrophin-releasing factor(12) . Additionally, an insect diuretic hormone receptor (13) and a partial genomic sequence from Caenorhabditis elegans(14) are homologous with the PTH/PTHrP receptor, indicating that this newly discovered family is widely conserved through evolution.

In primary and transformed cells derived from bone and kidney, the agonist-occupied PTH/PTHrP receptor activates multiple intracellular effectors, including adenylyl cyclase and phospholipase C(15, 16) . Activation of phospholipase C results in rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate, which generates two second messengers, inositol 1,4,5-triphosphate (IP(3)) and diacylglycerol(17, 18, 19) . IP(3) increases intracellular free calcium ([Ca]) by stimulating its release from the endoplasmic reticulum(20, 21, 22) , whereas diacylglycerol activates protein kinase C(23, 24, 25) . There also is evidence that PTH stimulates arachidonic acid metabolism(26) , changes membrane potentials(27) , and decreases intracellular pH(28) .

The goal of the present study was to analyze the role of the carboxyl-terminal tail of the PTH/PTHrP receptor in activating adenylyl cyclase and phospholipase C, by comparing the properties of R480 and R513, truncated rat PTH/PTHrP receptor mutants with carboxyl-terminal deletions of 111 and 78 amino acids, with those of the wild-type (WT) rat receptor, which is 591 amino acids in length. R480 has been shown to contain the minimal length necessary for full ligand binding, whereas R513 is the rat receptor equivalent of OK-H, a truncated version of the opossum PTH/PTHrP receptor (OK-O) studied previously (29) . When these three PTH/PTHrP receptors were expressed at closely similar cell surface densities, PTH stimulation of these truncated receptors strikingly increased cAMP accumulation without changing their capacities to stimulate polyphosphoinositide (PI) hydrolysis, compared with the WT receptor. The PTH/PTHrP receptor's carboxyl-terminal intracellular region contains domains that lower the binding affinity of the WT receptor for agonist, directly interact with or indirectly facilitates the interaction of the receptor with a pertussis toxin (PTX)-sensitive G protein that inhibits adenylyl cyclase, and decreases the efficacy with which the receptor couples to G


EXPERIMENTAL PROCEDURES

Materials

Rat PTH-(1-34)-NH(2) (rPTH) and bovine [Nle, Tyr]PTH-(1-34)NH(2) (NlePTH) were purchased from Bachem (Irvine, CA). Reagents of the highest purity available were obtained either from Sigma or Fisher. NaI (2125 Ci/mmol) for peptide and cAMP iodination, goat anti-rabbit I-IgG for receptor antibody binding studies, and S-dATP (1000-1500 Ci/mmol ) for sequencing were purchased from DuPont NEN. [^3H]Myoinositol (17.7 Ci/mmol) with PT6-271-polymer, which adsorbs decomposed radionuclides, was obtained from Amersham Corp. Restriction enzymes were purchased from U. S. Biochemical Corp., New England BioLab (Beverly, MA), and Promega (Madison, WI). DEAE-dextran for transfecting COS-7 cells was obtained from Pharmacia Biotech Inc. COS-7 cells were a generous gift from Dr. B. Seed, Laboratory of Molecular Biology, Massachusetts General Hospital (Boston, MA). Human [Tyr]PTHrP-(1-36)NH(2) (PTHrP) and oligonucleotides were synthesized by Dr. H. T. Keutmann of this Endocrine Unit.

Cell Culture

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech, Washington D. C.) supplemented with 7% fetal bovine serum (FBS, Hyclone, Logan, UT), 1.2 mM glutamine, and 4.5 g/liter of glucose at 37 °C in a humidified atmosphere containing 95% air and 5% CO(2). Medium was renewed every 3 days, and cells were passaged by trypsinization when near confluence.

Construction of Rat PTH/PTHrP Receptor Mutants

Fig. 1schematically depicts the 591-amino acid WT rat PTH/PTHrP receptor and the carboxyl-terminally truncated receptors R480 and R513. These truncated receptors were constructed using the polymerase chain reaction with the WT receptor serving as a template (30) . Antisense primers were designed to introduce stop codons followed by an XbaI site, leading to truncations of 111 (R480) and 78 (R513) amino acids of the carboxyl-terminal tail, respectively (GeneAmp, Perkin-Elmer), whereas a sense primer contained 24 nucleotides from the intact WT rat PTH/PTHrP receptor sequence with an NsiI site at 1398. Polymerase chain reaction products were first digested with NsiI and XbaI, purified on agarose gel, electroeluted, and then extracted with phenol/chloroform. The 2.2-kilobase pair WT receptor cDNA R15B-RI (31) in pcDNA1 (Invitrogen, San Diego, CA) was first digested with XbaI and then with NsiI, creating a vector with an NsiI site at position 1398 of the receptor and the XbaI site in the pcDNA1 polylinker region. The purified inserts then were ligated into the purified vector. The authenticity of the cloned mutant cDNAs was determined by restriction enzyme mapping and sequence analysis (32) of the nucleotides amplified by polymerase chain reaction and the adjacent sequences.


Figure 1: Schematic depiction of the 591-amino acid WT rat PTH/PTHrP receptor and R513 and R480, mutant receptors with 78- and 111-amino acid truncations of their carboxyl-terminal regions. Extracellular (EC) and intracellular (IC) regions are indicated



Transient Expression of PTH/PTHrP Receptors in COS-7 Cells

Receptor cDNAs in pcDNA1 were prepared by the CsCl method (33) and were introduced into COS-7 cells by the DEAE-dextran transfection method(34) . The quality and quantity of plasmid DNA was electrophoretically assessed on 0.8% agarose gel, after measuring OD at 260 nm. For transfections, a given amount of the plasmid DNA was diluted in 190 µl of phosphate-buffered saline (PBS, pH 7.42) and then this mixture was further diluted in 760 µl of 10 mg/ml DEAE-dextran solution. Cells, in 150-mm dishes (approximately 10^7 cells), were first gently washed twice with prewarmed PBS and exposed to 19 ml of DMEM containing 10% Nuserum (Life Technologies, Inc.) and 100 µM chloroquine. The transfection mixture of plasmids in the DEAE-dextran solution was then added to the cells. The transfection mixture was removed after a 3-h incubation at 37 °C and then the cells were exposed to PBS containing 10% dimethyl sulfoxide (Me(2)SO) at room temperature for 2 min. The cells were then cultured in DMEM supplemented with 7% FBS for an additional 24 h and replated into 24-, 12-, or 6-well plates at a density of 50-100,000 cells/cm^2. Cells were studied 60-72 h after transfection.

Radioreceptor Assay

Transfected cells grown in 24-well plates were washed twice with 1 ml of binding buffer, pH 7.7, containing 100 mM NaCl, 5 mM KCl, 2 mM CaCl(2), 50 mM Tris, 5% heat-inactivated horse serum (Life Technologies, Inc.), and 0.5% FBS. For radioreceptor assays, cells were incubated with I-[Tyr]PTHrP-(1-36)-NH(2)



(PTHrP) 100,000 cpm/500 µl/well in binding buffer in the absence or presence of unlabeled rPTH-(1-34) (10 to 10M) at 16 °C for 4 h. In some experiments, we used bovine I-[Nle,Tyr]PTH-(1-34)-NH(2) instead of PTHrP, since we found no significant difference in results of experiments using these two ligands(35) . Cells were then processed, and bound radioactivity was measured, as described previously(22, 35) . Cells in two wells from each plate were suspended by trypsinization and cell number established using a hemocytometer. Specific binding was determined by subtracting binding in the presence of 10M rPTH from total binding.

Quantification of Expressed PTH/PTHrP Receptors

Receptor number per transfected cell and their affinities were determined by Scatchard analysis(36) , after correcting for transfection efficiency, and receptor density was confirmed using a sheep antireceptor antiserum, G48, which was developed against a synthetic 18-amino acid peptide (DKGWTPASTSGKPRKEKA) of the rat PTH/PTHrP receptor's extracellular amino-terminal region. The antiserum was purified by affinity chromatography with antigen immobilized to Sepharose 4B by the CNBr method(37) . Transfected cells, grown in 24-well plates, were washed three times with PBS, pH 7.4, containing 5% FBS and then incubated at room temperature for 1 h in PBS in the presence of G48 (0.5 µg/250 µl/well, 1:500 dilution) or preimmune sheep serum (same dilution as G48). Cells were then washed thoroughly (four times) with 5% FBS/PBS, incubated first for 1 h at room temperature with affinity-purified rabbit anti-sheep IgG (H+L) (Kirkland & Perry Laboratories, Inc., Gaithersburg, MD) and then with 200-300,000 cpm/250 µl/well of goat anti-rabbit I-IgG. After 1 h, the cells were washed three times with PBS, solubilized in 1 N NaOH, and the radioactivity was counted.

Quantification of Transfected COS-7 Cells by Immunofluorescence

Transfected cells were treated exactly as described above, but rather than adding goat anti-rabbit I-IgG, we added mouse anti-rabbit IgG conjugated with fluorescein isothiocyanate (Sigma) (22 °C, 1 h) and fixed the cells with 2% paraformaldehyde, after washing them three times with 5% FBS/PBS. The number of transfected, receptor-bearing cells was counted using fluorescence microscopy (Zeiss, Axiouscope, Oberkoken, Germany), and the total number of cells was determined under bright field. Five different fields in each of three wells per group were counted.

Measurement of Intracellular cAMP Production

Transfected cells were preincubated in serum-free DMEM containing 0.1% BSA, 10 mM Hepes, pH 7.42, and 1 mM 3-isobutyl-1-methylxanthine (IBMX) at room temperature for 10 min and further incubated at 37 °C for an additional 15 min after adding agonist or vehicle. Cyclic AMP accumulation was determined after a 15-min incubation with rat PTH-(1-34) (rPTH), because PTH (100 nM) produced a linear increase in intracellular cAMP concentration in COS-7 cells for up to 30 min in the presence of 1 mM IBMX (Fig. 2A). Basal cAMP levels were measured in transfected cells that were incubated at 37 °C for 15 min in the presence of 1 mM IBMX. Reactions were terminated by aspirating the medium, washing the cells with ice-cold PBS, and freezing the cells in 500 µl of 50 mM HCl. Cells were stored at -80 °C until intracellular cAMP was measured by radioimmunoassay, as described previously(23) . Hormone-stimulated cAMP accumulation is expressed after subtracting basal values.


Figure 2: Time course of PTH-stimulated cAMP accumulation and PI hydrolysis in COS-7 cells expressing the WT rat PTH/PTHrP receptor. COS-7 cells (10^7 cells/150-mm dish) were transfected with 5 µg of DNA, replated into 24-well plates, and incubated with PTH (100 nM) or vehicle alone, under conditions described under ``Experimental Procedures.'' Intracellular cAMP accumulation (A) and (B) IP(1) (circle), IP(2) (bullet), and IP (up triangle) were measured at 37 °C from 1 to 60 min. Experiments were conducted in triplicate, and results were calculated after subtraction of basal values, which were obtained in the absence of hormone. Data (mean ± S.E.) are from one of two experiments, both of which had closely similar results. When not shown, S.E. values are so small that they fall within the symbols.



Measurement of PI Hydrolysis

Transfected cells, grown in six-well plates, were labeled at 37 °C for 8-12 h with 3 µCi/ml of [^3H]inositol in serum- and inositol-free DMEM (Life Technologies, Inc.) supplemented with 0.1% BSA. (Preliminary studies had shown that the inositol pool of the COS-7 cells was labeled to equilibrium with [^3H]myoinositol by 8 h.) After cells were washed twice with prewarmed serum- and inositol-free DMEM containing 20 mM LiCl and 0.1% BSA, they were treated with rPTH (10 to 10M) or with v ehicle alone (10 mM acetic acid, basal values) at 37 °C for an additional 30 min. PTH (100 nM) produced time-dependent increases in IP(1) and IP(2) accumulation that were linear for at least 1 h (Fig. 2B). However, since the IP(3) production rate was linear only for the first 30 min, we used 30-min incubations for all experiments. The reaction was terminated by aspirating the incubation medium and adding 1 ml of ice-cold 5% trichloroacetic acid. After extracting the trichloroacetic acid with water-saturated diethyl ether (three times) and adjusting the pH of the solution to 7.4, the solutions were chromatographed on AG 1 times 8 anion-exchange columns (formate form, 100-200 mesh, Bio-Rad). After free [^3H]inositol and glycerophosphate inositol fractions were eluted with 10 ml of 10 mM inositol and 8 ml of 5 mM Borax, 60 mM sodium formate, respectively, the IP(1), IP(2), and IP(3) fractions were collected by sequential elution with 8, 10, and 10 ml of 0.2, 0.5-0.7, and 1.05 M ammonium formate, 0.1 M formic acid, respectively(17, 21) . IP(2) was first sequentially eluted with 1 ml each of 0.5, 0.55, 0.6, and 0.65 M and 6 ml of 0.7 M ammonium formate, 0.1 M formic acid; these eluents were then pooled as the ``IP(2)'' fraction. This modification provides better resolution of IP(2) from IP(3). Radioactivity in 1 ml of each fraction was counted with a liquid scintillation counter (Beckman, model LS 6000IC). Hormone-stimulated PI hydrolysis was determined after subtracting basal values from the values obtained in the presence of hormone. Total IPs are the sum of IP(1), IP(2), and IP(3).

Statistical Analysis

Means ± S.E. of duplicate or triplicate samples were calculated from 2-17 independent experiments. Data were analyzed using one way analysis of variance and the Scheffe-F test to determine significance.


RESULTS

We first characterized the full-length WT receptor's capacities to bind ligands and antireceptor antibody and to stimulate adenylyl cyclase and phospholipase C. In COS-7 cells transiently expressing the WT receptor (2-3 times 10^6 receptors/transfected cell), the apparent K(d) for I-PTHrP binding was 2.1 ± 0.3 nM, whereas EC values for cAMP accumulation and PI hydrolysis were 0.3 ± 0.1 nM and 4.0 ± 0.2 nM, respectively (Fig. 3; since the PI hydrolysis response was not quite maximal at 10M PTH, this EC is an approximation). Significant stimulation of cAMP accumulation was detected in cells treated with as low as 10M PTH, a concentration that did not detectably displace the radioactive ligand. PTH-stimulated PI hydrolysis, however, was substantially less sensitive than PTH-stimulated cAMP accumulation; it was first detected at ligand concentrations between 10 and 10M. When the two second messenger responses are normalized by setting maximal responses to 100%, lower levels of receptor occupancy elicited relatively greater increases in cAMP accumulation, compared with the level of receptor occupancy needed for similar increases in PI hydrolysis.


Figure 3: Dose-response relationship of PTH binding and PTH-stimulated cAMP accumulation and PTH-stimulated PI hydrolysis in COS-7 cells expressing the WT rat PTH/PTHrP receptor. COS-7 cells (10^7/150-mm dish) were transfected with 5 µg of DNA, replated, and incubated with PTH (10 to 10M) or vehicle alone, under conditions described under ``Experimental Procedures.'' Radioreceptor assays were performed with I-PTHrP, at 16 °C for 4 h. Specific binding (circle) was calculated by subtracting binding in the presence of PTH (1 µM) from total binding and expressed as a percentage of specific binding in the absence of unlabeled rPTH; cyclic AMP accumulation (bullet) and total IPs (up triangle) generated were measured after 15 and 30 min, respectively, and are expressed as a percentage of maximally stimulated values. Data (mean ± S.E.) are from seven radioreceptor assays, and three bioassays each for cAMP and IP metabolites, all of which were performed in triplicate. When not shown, S.E. values are so small that they fall within the symbols.



Cell surface expression markedly differed among the mutant and WT receptors, when the same amount (5 µg) of plasmid DNA was used for transfection. Since we sought to study the properties of these receptors at similar expression levels, we first determined the levels of cell surface expression for each receptor, after transfecting COS-7 cells with varying amounts of plasmid DNA (0.05-5 µg of WT and 0.1-10 µg of R513 and R480). The number of PTH binding sites per cell, as calculated from Scatchard analysis, was dependent on the amount of introduced plasmid DNA, but markedly differed among these receptor constructs (Fig. 4A). Levels of receptor expression comparable with those obtained with the WT receptor could only be achieved by transfecting the COS-7 cells with higher amounts of either truncated receptor construct.


Figure 4: The relationship between the amount of plasmid DNA used for transfecting COS-7 cells and the number of expressed receptors. COS-7 cells were transfected with varying amounts of plasmid DNA (0.05-10 µg) containing WT (circle) or truncated mutant receptors R513 (bullet) and R480 (up triangle). A, the number of receptors per transfected cell, as calculated by Scatchard analysis. B, the specific I-labeled goat anti-rabbit IgG bound was determined, after the cells were incubated sequentially with sheep anti-receptor antibody, G48, and rabbit anti-sheep IgG (see ``Experimental Procedures''). Data (mean ± S.E.) are from one of four representative experiments, all of which were performed in triplicate. When not shown, S.E. values are so small that they fall within the symbols.



To further characterize the relationship between cell surface expression of the three receptors and the amount of the transfected plasmid DNA, we assessed whether changes in cell surface expression were due to variation in the number of cells expressing receptors or to variations in the number of receptors expressed per cell. When COS-7 cells were transfected with 0.1 or 5 µg of plasmid DNA containing the WT receptor construct, there was no significant difference in the percentage of COS-7 cells that stained positively by immunofluorescence (Table 1). In all experiments, the transfection efficiency for the WT receptor construct in COS-7 cells was consistently 17-20%, regardless of the amount of DNA used, and was indistinguishable from the transfection efficiency for either mutant receptor construct (Table 1). Thus, the amount of cDNA introduced determined PTH/PTHrP receptor's cell surface expression per cell, but did not influence the number of transfectable cells in this transient expression system. To match the expression level of each receptor, we varied the amount of DNA used for transfection, while maintaining a ratio of plasmid DNA for R591/R513/R418 of 1/25/50, respectively. The results from G48 antireceptor antibody binding confirmed observations based on Scatchard analysis (Fig. 4B).



Radioreceptor assays were conducted in triplicate for each construct, over a range of 5 times 10^4 to 10^6 receptors/transfected cell. Scatchard analysis showed that the apparent K(d) values of rPTH-(1-34) for WT, R513, and R480 receptors were 1.81 ± 0.19 nM (n = 17), 1.24 ± 0.12 nM (n = 11), and 0.48 ± 0.05 nM (n = 13), respectively, and were consistent within each group over this level of receptor expression. All were statistically different from each other: WT versus R513 (p < 0.05), WT versus R480 (p < 0.001), R513 versus R480 (p < 0.01).

Next, we correlated the level of receptors expressed with the activated receptor's capacity to stimulate cAMP accumulation and PI hydrolysis. Ligand binding, cAMP accumulation, and PI hydrolysis were always compared in cells from the same transfection. PTH (100 nM)-stimulated PI hydrolysis in COS-7 cells expressing WT, R513 (Fig. 5A), and R480 receptors (Fig. 6A) was linearly dependent upon the level of receptor expression and was indistinguishable among the WT and the two truncated receptors ( Fig. 5and Fig. 6). In contrast, although PTH-stimulated cAMP accumulation also correlated with the level of receptor expression, it was 2-4- and 4-6-fold higher in the cells expressing R513 (Fig. 5B) and R480 (Fig. 6B), respectively, compared with the WT receptor, at all levels of receptor expression.


Figure 5: The signal transduction properties of the WT rat PTH/PTHrP receptor compared with R513, as a function of the number of receptors expressed. COS-7 cells were transfected with varying amounts of plasmid DNA containing either WT (circle) or mutant R513 (bullet) receptors, radioreceptor assays were then performed, and the number of receptors expressed per transfected cell was determined by Scatchard analysis. The capacity of COS-7 cells expressing these receptors to generate IP metabolites (A) and to increase intracellular cAMP (B) in response to treatment with PTH (100 nM) then was determined, as described under ``Experimental Procedures.'' Data (mean ± S.E.) are from one of three representative experiments, all of which were performed in triplicate. When not shown, S.E. values are so small that they fall within the symbols.




Figure 6: The signal transduction properties of the WT rat PTH/PTHrP receptor compared with R480, as a function of the number of receptors expressed. COS-7 cells were transfected with varying amounts of plasmid DNA containing either WT (circle) or mutant R480 (bullet) receptors, radioreceptor assays were then performed, and the number of receptors expressed per transfected cell was determined by Scatchard analysis. The capacity of COS-7 cells expressing these receptors to generate IP metabolites (A) and to increase intracellular cAMP (B) in response to treatment with PTH (100 nM) then was determined, as described under ``Experimental Procedures.'' Data (mean ± S.E.) are from one of three representative experiments, all of which were performed in triplicate. When not shown, S.E. values are so small that they fall within the symbols.



We then assessed the dose-response relationships for PTH-stimulated cAMP accumulation in COS-7 cells expressing comparable levels of WT, R513, and R480 receptors. To achieve these levels, we transfected cells with 0.1, 2.5, and 5.0 µg of plasmid DNA containing WT, R513, and R480 receptor constructs, respectively. These transfections resulted in expression of 620,000, 505,000, and 520,000 per cell for WT, R513, and R480 receptors, respectively, as determined by Scatchard analysis. Maximal cAMP stimulation was 2- and 6-fold higher in COS-7 cells expressing R513 and R480, respectively, than in those expressing the WT receptor (Fig. 7); however, the EC values of PTH-stimulated cAMP accumulation were similar for the three constructs.


Figure 7: PTH-stimulated intracellular cAMP response of COS-7 cells expressing comparable levels of WT and mutant receptors. PTH-stimulated intracellular cAMP accumulation was determined, over a hormonal concentration range of 10 to 10M, in COS-7 cells transfected with varying amounts of plasmid DNA (see ``Experimental Procedures'' for details). As determined by Scatchard analysis, similar levels of receptors per transfected cell were expressed (WT, 620,000; R513, 505,000; R480, 520,000), when COS-7 cells were transfected with 0.1, 2.5, and 5 µg of plasmid DNA, respectively. Data (mean ± S.E.) are from one of three representative experiments, all of which were performed in triplicate. When not shown, S.M. values are so small that they fall within the symbols.



We and others have reported previously that pretreatment of a rat osteoblast-like osteosarcoma cell line, ROS 17/2.8(38, 39) , and human osteoblasts in primary culture (40) with PTX augmented PTH-stimulated cAMP accumulation, without any significant alteration of the basal cAMP levels. These results suggested coupling of the native PTH receptor to a PTX-sensitive inhibitory G protein, putatively a member of the G(i) family. Since truncated receptors had higher maximal PTH-stimulated cAMP accumulation than WT receptors, we tested the hypothesis that a putative PTX-sensitive G(i) protein might couple to the WT receptor, but not to the truncated receptors. COS-7 cells expressing similar levels of WT and truncated (R513 or R480) PTH/PTHrP receptors (5-6 times 10^5 binding sites/cell) were treated with 100 ng/ml PTX for 14 h before stimulating the cells with 10 to 10M PTH. PTX augmented PTH-stimulated cAMP production by 2-fold at all concentrations of PTH in cells expressing WT receptors, but it failed to alter PTH-stimulated cAMP accumulation in cells expressing either mutant receptor (Fig. 8).


Figure 8: The effect of PTX pretreatment on PTH-stimulated cAMP accumulation in COS-7 cells expressing WT (left), R513 (middle), and R480 (right) receptors. COS-7 cells were transfected with varying amounts of plasmid DNA containing either WT or the two mutant receptors, radioreceptor assays were then performed, and the number of receptors expressed per transfected cell was determined by Scatchard analysis. Cells expressing similar numbers of WT, R513, and R480 receptors (5-6 times 10^5 binding sites/cell) were treated with PTX (bullet, 100 ng/ml) or vehicle alone (circle) for the last 14 h in serum-free DMEM medium containing 0.1% BSA, prior to stimulation with PTH (10 to 10M). Data (mean ± S.E.) are from one of two representative experiments, both of which were performed in triplicate. When not shown, S.E. values are so small that they fall within the symbols.




DISCUSSION

Our most striking observation is that rat PTH/PTHrP receptors with truncated carboxyl-terminal, intracellular regions signal adenylyl cyclase with markedly higher efficacy, compared with the WT receptor, but have indistinguishable capacities to hydrolyze PI.

We initially noted lower ligand binding in cells transfected with R480 and R513, compared with the WT receptor, when the same amount of DNA was used for transfection. Because the properties of WT and truncated receptors might be influenced by the levels at which they were expressed, we first sought conditions that matched levels of functional receptors. These preliminary studies demonstrated that the number of receptors expressed per transfected cell was markedly influenced by both the amount of plasmid DNA and by the specific construct used for transfection, but that the percentage of transfected cells was independent of either parameter; that is, each transfected COS-7 cell expressed WT receptors more efficiently over a broad range of added DNA, compared with their expression of either R480 and R513. Careful control of the conditions for COS-7 cell's incubation and transfection and of the amount of plasmid DNA used for transfection enabled us to compare the properties of the truncated and WT receptors at closely similar levels of receptors per cell and over a relatively wide range of expressed receptors.

The magnitude of both maximal PTH-stimulated cAMP accumulation and PI hydrolysis depended on the level of cell surface expression in COS-7 cells. The rates of PI hydrolysis were indistinguishable between either truncated mutant and WT receptor when cells expressing matched levels of the three receptors were treated with 100 nM PTH, over a wide range of receptor levels. In contrast, R480 and R513 increased cAMP accumulation by 5-6- and 2-3-fold, respectively, compared with the WT receptor expressed at comparable levels, when treated with the same dose of hormone. These data confirm and extend those of an earlier observation from our laboratory, which demonstrated that PTH-stimulated increases in intracellular [Ca] and increases in the rates of PI hydrolysis correlated directly with the number of PTH/PTHrP receptors stably expressed in LLC-PK1 cells, a porcine kidney cell line without endogenous PTH/PTHrP receptors(22, 41) .

In a previous study(29) , we had noted that a carboxyl-terminally truncated opossum kidney PTH/PTHrP receptor (OK-H) that contains 508 of the 585 amino acids of the full-length receptor (OK-O), and is the opossum homolog of R513, seemed to have unimpaired capacity to activate adenylyl cyclase, but reduced capacity to stimulate the rate of PI hydrolysis. In recent experiments in which receptor expression was carefully controlled, however, we found, instead, that OK-H had an unimpaired capacity to stimulate PI hydrolysis and enhanced capacity to stimulate cAMP accumulation (data not shown). We had misinterpreted our early experiments because we had failed to rigorously control for the number of receptors expressed per cell.

Our observation that the intact, cloned PTH/PTHrP receptor interacts with a PTX-sensitive G protein linked to inhibition of adenylyl cyclase extends earlier observations we and others have made in studies of endogenous receptors on ROS 17/2.8 cells (38, 39) and primary cultures of human osteoblasts(40) , where PTX treatment enhanced intracellular cAMP accumulation, without altering basal levels. Since the PTX effect was seen only with the WT receptor, the receptor's interaction with this inhibitory G protein must rely, directly or indirectly, on the sequence between residues 513 and 591. Future efforts are needed to further identify the domain(s) involved in mediating this function.

Other aspects of our observations also will require additional experiments before they are fully understood. For example, the higher efficacy (maximal cAMP responsiveness) with which R480 activates adenylyl cyclase, compared with the WT receptor, can be attributed in part to the absence of a site that enables the PTH/PTHrP receptor to couple to a PTX-sensitive G protein which inhibits adenylyl cyclase and which is present between residues 513 and 591 of the receptor's sequence. Other factors, however, must also be involved in modulating this response, because PTH treatment of R513 elicits a maximal cAMP response that is lower than the response of R480, although both mutant receptors are insensitive to PTX, but higher than the response of the WT receptor, which is sensitive to PTX.

Second, the higher binding affinities with which R480 and R513 bind agonist, compared with the WT receptor, would predict lower EC values of both adenylyl cyclase and phospholipase C responses, if the increased binding energy were associated with a conformation that activated G proteins. This was not observed with either effector response; neither the EC nor the maximal phospholipase C response was modified, nor was any change noted in the EC of the adenylyl cyclase response, although the strikingly higher maximal cAMP accumulation might have made it difficult to appreciate any change. Taken together, these data suggest that the affinity of ligand binding to the these receptors can be dissociated from activation of the two effector pathways. This appears to clearly be the the case with activation of phospholipase C. With respect to activation of adenylyl cyclase, these truncated PTH/PTHrP receptors appear to have higher capacities to increase intracellular cAMP accumulation, compared with the WT receptor. It is difficult to relate this property directly to the changes in ligand binding affinity, however, because no change in the EC values was noted. Additionally, the different affinities with which these two truncated receptors bind agonist, when expressed in COS-7 cells, suggest that multiple intracellular receptor sequences influence this property. Interestingly, Parker and Ross (42) showed that truncated avian beta-adrenergic receptors activated adenylyl cyclase with higher efficacy than the full-length receptor. Although these truncated receptors had modestly increased affinity for ligands, the influence of PTX on the adenylyl cyclase response was not assessed.

Studies of PTH/PTHrP receptors transiently expressed in COS-7 cells, however, have limitations as well as certain advantages. They allow relatively rapid comparisons of different receptors at various levels of expression, which can be rigorously controlled at levels above 10^4 receptors/transfected cell, and they provide a system that allows the properties of these receptors to be assessed independently without complicating variables, such as clonality that influence stably expressed receptors. On the other hand, receptor expression is difficult to control at more modest levels. Furthermore, the signaling properties of the PTH/PTHrP receptor necessarily depend on the particular complement of G proteins and other cytoplasmic factors present in COS-7 cells. Studies in which PTH/PTHrP receptors are characterized in the context of a cellular milieu other than COS-7 cells, therefore, are likely to provide additional information.

These results highlight the functional importance of the carboxyl-terminal region of the PTH/PTHrP receptor, as manifested mainly by the striking discordance between the increased PTH-stimulated cAMP accumulation and the unchanged response in PTH-stimulated PI hydrolysis, when the properties of mutant PTH/PTHrP receptors with deletions of this region are compared with those of the WT receptor. Sequences in the PTH/PTHrP receptor's carboxyl-terminal intracellular region have multiple influences on receptor function: they lower the apparent affinity of the WT receptor for agonist; contain domains distal to amino acid 513 that directly interact with, or indirectly facilitate its interaction with, a PTX-sensitive G protein, presumably a member of the G(i) family that inhibits adenylyl cyclase; and they decrease the efficacy (maximal agonist-stimulated cAMP accumulation) with which the receptor interacts with G(s).


FOOTNOTES

*
This work was supported by National Institutes of Health Individual National Research Service Fellowship 1-F32-DK08751 (to A. Iida-Klein) and National Institutes of Health Grants DK47034 and DK11794. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-3966; Fax: 617-726-7543.

(^1)
The abbreviation s used are: PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; IP(1), inositol phosphate; IP(2), inositol bisphosphate; IP(3), inositol triphosphate; IPs, inositol phosphates; WT, wild-type; PI, polyphosphoinositide; PTX, pertussis toxin; rPTH, rat PTH-(1-34); DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine.


ACKNOWLEDGEMENTS

We thank Dr. Toshiro Usa for his assistance in preparation of figures and statistical analyses and James D. Deeds and Alicia Lee for their technical help.


REFERENCES

  1. Jüppner, H., Abou-Samra, A. B., Freeman, F. M., Kong, X.-F., Schipani, E., Richards, J., Kolakowski, L. F., Jr., Hock, J., Potts, J. T., Jr., Kronenberg, H. M., and Segre, G. V. (1991) Science 254, 1024-1026 [Medline] [Order article via Infotrieve]
  2. Abou-Samra, A. B., Jüppner, H., Force, T., Freeman, F. M., Kong, X.-F., Schipani, E., Urena, P., Richards, J., Bonventre, J. V., Potts, J. T., Jr., Kronenberg, H. M., and Segre, G. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2732-2736 [Abstract]
  3. Lin, H. Y., Harris, T. L., Flannery, M. S., Aruffo, A., Kaji, E. H., Gorn, A., Kolakowski, L. F., Jr., Lodish, H. F., and Goldring, S. R. (1991) Science 254, 1022-1024 [Medline] [Order article via Infotrieve]
  4. Ishihara, T., Nakamura, S., Kaziro, Y., Takahashi, T., Takahashi, K., and Nagata, S. (1991) EMBO J. 10, 1635-1641 [Abstract]
  5. Jelinek, L. J., Lok, S., Rosenberg, G. B., Smith, R. A., Grant, F. J., Biggs, S., Bensch, P. A., Kuijper, J. L., Sheppard, P. O., Sprecher, C. A., O'Hara, P. J., Foster, D., Walker, K. M., Chen, L. H. J., McKerman, P. A., and Kindsvogel, W. (1993) Science 259, 1614-1616 [Medline] [Order article via Infotrieve]
  6. Thorens, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8641-8645 [Abstract]
  7. Mayo, K. E. (1992) Mol. Endocrinol. 6, 1734-1744 [Abstract]
  8. Ishihara, T., Shigemoto, R., Mori, K., and Nagata, S. (1992) Neuron 8, 811-819 [Medline] [Order article via Infotrieve]
  9. Lutz, E. M., Sheward, W. J., West, K. M., Morrow, J. A., Fink, G., and Harmer, A. J. (1993) FEBS Lett. 334, 3-8 [CrossRef][Medline] [Order article via Infotrieve]
  10. Wank, S. A., and Pisegna, J. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6345-6349 [Abstract]
  11. Usdin, T. B., Mesey, E., Button, D. C., Brownstein, N. J., and Bonner, T. J. (1993) Endocrinology 133, 2861-2870 [Abstract]
  12. Chen, R., Lewis, K. A., Perrin, M. H., and Vale, W. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8967-8971 [Abstract]
  13. Reagan, J. D. (1994) J. Biol. Chem. 269, 9-12 [Abstract/Free Full Text]
  14. Sulston, J., Du, Z., Thomas, K., Wilson, R., Hillier, L., Staden, R., Halloran, N., Green, P., Thierry-Mirg, J., Qiu, L., Dear, S.,, Coulson, M., Craxton, M., Durbin, R. K., Berks, M., Metzstein, M., Hawkins, T., Inscough, R. A., and Waterston, R. (1992) Nature 356, 37-41 [CrossRef][Medline] [Order article via Infotrieve]
  15. Kronenberg, H. M., Bringhurst, F. R., Nussbaum, S., J ü ppner, H., Abou-Samra, A.-B., Segre, G., and Potts, J. T., Jr. (1993) Handbook of Experimental Pharmacology , Vol. 107, pp. 507-567, Springer-Verlag, Berlin
  16. Jüppner, H. (1994) Curr. Opin. Nephrol. Hypertens. 3, 371-378 [Medline] [Order article via Infotrieve]
  17. Berridge, M. J., Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984) Biochem. J. 222, 195-201 [Medline] [Order article via Infotrieve]
  18. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  19. Civetelli, R., Reid, I. R., Westbrook, S., Avioli, L. V., and Hruska, K. A. (1988) Am. J. Physiol. 253, E660-E667
  20. Yamaguchi, D. T., Hahn, T. J., Iida-Klein, A., Kleeman, C. R., and Muallem, S. (1987) J. Biol. Chem. 262, 7711-7718 [Abstract/Free Full Text]
  21. Iida-Klein, A., and Hahn, T. J. (1991) Endocrinology 129, 1016-1024 [Abstract]
  22. Bringhurst, F. R., Jüppner, H., Guo, J., Urena, P., Potts, J. T., Jr., Kronenberg, H. M., Abou-Samra, A. B., and Segre, G. V. (1992) Endocrinology 132, 2090-2098 [Abstract]
  23. Abou-Samra, A. B., Jüppner, H., Westberg, D., Potts, T. J., Jr., and Segre, G. V. (1989) Endocrinology 124, 1107-1113 [Abstract]
  24. Iida-Klein, A., Varlotta, V., and Hahn, T. J. (1989) J. Bone Miner. Res. 4, 767-774 [Medline] [Order article via Infotrieve]
  25. Nishizuka, Y. (1986) Science 233, 305-312 [Medline] [Order article via Infotrieve]
  26. Feyen, J. H. M., Van Der Wilt, G., Moonen, P., DiBon, A., and Nijiweide, P. J. (1984) Prostaglandins 28, 269-281
  27. Edelman, A., Fritsch, J., and Balsam, S. (1986) Am. J. Physiol. 251, C483-C490
  28. Reid, I. R., Civetelli, R., Avioli, L. V., and Hruska, K. A. (1987) Am. J. Physiol. 225, E9-E15
  29. Segre, J. V., Abou-Samra, A. B., Jüppner, H., Schipani, E., Force, T., Urena, P., Freeman, M., Kong, X. F., Kolakowski, L. F., Hock, J., Bonventre, J., Potts, J. T., Jr., and Kronenberg, H. M. (1992) J. Endocrinol. Invest. 15, 11-17 [Medline] [Order article via Infotrieve]
  30. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Science 243, 217-219 [Medline] [Order article via Infotrieve]
  31. Jüppner, H., Schipani, E., Bringhurst, F. R., McClure, I., Keutmann, H. T., J., Potts, J. T., Jr., Kronenberg, H. M., Abou-Samra, A. B., Segre, G. V., and Gardella, T. J. (1994) Endocrinology 134, 879-884 [Abstract]
  32. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448 [Medline] [Order article via Infotrieve]
  33. Sambrook, J., Fritsch, E. F., and Maniatas, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., pp. 1.41-1.50, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Lopata, M. A., Cleveland, D. W., and Solner-Webb, B. (1984) Nucleic Acids Res. 12, 5707-5711 [Abstract]
  35. Jüppner, H., Abou-Samra, A. B., Uneno, S., Gu, W. X., Potts, J. T., Jr., and Segre, G. V. (1988) J. Biol. Chem. 263, 8557-8560 [Abstract/Free Full Text]
  36. Bylund, D. B., and Yamamura, H. I. (1990) in Methods for Receptor Binding (Yamamura, H. I., Enna, S. J., and Kuhar, M. J., eds) pp. 1-35, Raven Press, Ltd., New York
  37. Nagai, K., and Thogersen, H. C. (1984) Nature 309, 810-812 [Medline] [Order article via Infotrieve]
  38. Abou-Samra, A.-B., Jüppner, H., Potts, J. T., Jr., and Segre, G. V. (1989) Endocrinology 125, 2594-2599 [Abstract]
  39. Pines, M., Santora, A., Gierschik, P., Menczel, J., and Spiegel, A. (1986) Bone Miner. 1, 15-20 [Medline] [Order article via Infotrieve]
  40. Iida-Klein, A., Varlotta, V., Yee, D. C., Kobayashi, R., and Hahn, T. J. (1989) J. Bone Miner. Res. 4, Suppl. 1, 879
  41. Guo, J., Abou-Samra, A. B., and Bringhurst, F. R. (1993) J. Bone Miner. Res. 8, Suppl. 1, S-176
  42. Parker, E. M., and Ross, E. M. (1991) J. Biol. Chem. 266, 9987-9996 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.