Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTH-related protein receptor result in selective loss of PTH-stimulated phospholipase C activity.

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 +/- 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Galphaq-subunits for transducing the receptor signal, Galphaq was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Galphaq. In contrast, PTH generated 2- and 2. 5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Galphaq. Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, alpha-subunits of the Gq family, rather than betagamma-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate co-transport in LLC-PK1 cells.

Signals from the parathyroid hormone (PTH) 1 /PTH-related peptide (PTHrP) receptor are transduced by G proteins. The lack of sequence homology between PTH/PTHrP receptors and all but a few of the other G protein-coupled receptors, however, justifies classifying them as members of a distinct family (1), which includes two mammalian receptors each for PTH or PTHrP (2,3), vasoactive intestinal peptides (4,5), and corticotrophin-releasing hormone (6), and receptors for secretin (7), calcitonin (8), glucagon-like peptide 1 (9), growth hormonereleasing hormone (10), glucagon (11), pituitary adenylyl cyclase-activating peptide (12), gastric inhibitory peptide (13), and an orphan receptor in brain similar to the calcitonin receptor (14). Receptors from this family are not limited to vertebrates, as two insect diuretic hormone receptors (15) and a partial genomic clone from Caenorhabditis elegans (16) also are homologous. Rat, opossum, and human PTH/PTHrP receptors activate multiple signal transduction pathways that include both adenylyl cyclase and phospholipase C (PLC) (1,17); they bind two ligands, PTH and PTHrP, with nearly equal efficacy (1,2), and they are widely distributed in various tissues including the major PTH targets, kidney and bone (18,19). Little, however, is known concerning the structural features of PTH/PTHrP receptors that enable them to activate these multiple effectors.
We previously showed that the rat PTH/PTHrP receptor with truncations of its C-terminal intracellular tail had markedly enhanced capacity to increase intracellular cAMP, but unaltered capacity to generate inositol phosphates (IPs), when expressed at levels comparable with the WT receptor (20). These observations extended those of Nissenson et al. (21), who showed that the PTH/PTHrP receptor's C-terminal tail could be deleted or mutated without major loss in its capacity to increase intracellular cAMP. PTH-stimulated increases in cAMP and IPs are both dependent on the level of receptors expressed, but we showed that adenylyl cyclase responsiveness is elicited at lower PTH concentrations and when fewer receptors are expressed, compared with the PLC response (20,22).
To determine portions of the PTH/PTHrP receptor important for activation of PLC, we first constructed receptors with random clustered mutations in the putative second intracellular (2i) loop of the rat PTH/PTHrP receptor and screened them for ligand binding and adenylyl cyclase stimulating properties. Then, mutant receptors whose maximal radioligand binding and maximal PTH-stimulated cAMP response were at least 50% as great as those of the WT receptor were further characterized for their capacity to stimulate PLC, as assessed by measuring PTH-stimulated accumulation of IPs. This initial screening identified a receptor containing a random cassette mutation, DSEL (amino acids 317-320) in the 2i loop rather than the WT sequence EKKY, which had little or no capacity to stimulate PLC, but its capacity to bind the ligand and to stimulate adenylyl cyclase was unimpaired.
We then systematically examined which amino acid in this 2i loop sequence accounted for PLC activation by testing the functional properties of PTH/PTHrP receptors with single-and double-point mutations of the WT sequence, EKKY, and singlepoint "revertants" of receptors with DSEL mutant cassette. The studies reported here highlight the critical role of a portion of the receptor's 2i loop, especially of lysine at position 319, for selective coupling of the PTH/PTHrP receptor to member(s) of the G q family of ␣-subunits that results in PLC activation. Furthermore, we extend our previous observations to show that sodium-dependent phosphate co-transport in LLC-PK1 cells that stably express PTH/PTHrP receptors is independent of cAMP and that it is mediated through a PLC pathway. Cell Culture-COS-7 and LLC-PK1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech, Washington, D.C.) supplemented with 7% fetal bovine serum (Sigma), 1.2 mM glutamine, and 4.5 g/liter glucose at 37°C, in a humidified atmosphere containing 95% air and 5% CO 2 . HEK293 cells were grown in minimum essential Eagle's medium supplemented with 10% horse serum (Life Technologies, Inc.). Medium was replenished every 2-3 days, and cells were passaged when nearly confluent.

Materials-Rat
Construction of Mutant Rat PTH/PTHrP Receptors-Mutant rat PTH/PTHrP receptors were constructed by site-directed mutagenesis, using a minor modification of the method of Kunkel et al. (23). Oligonucleotides were designed to either randomly mutate amino acids in the WT receptor's 2i loop (amino acids 316 -320, SEKKY) or to modify individual amino acids between 317 and 320. The codons targeted for mutagenesis were replaced by NNG/C, where N is an equal mixture of all four nucleosides and G/C is an equal mixture of G and C. Briefly, the mutant oligonucleotides in a sense orientation were phosphorylated by T4 polynucleotide kinase at 37°C for 45 min in a buffer (pH 7.8), containing Tris (100 mM), dithiothreitol (10 mM), ATP (0.4 mM), and MgCl 2 (10 mM), and the mixture then was heated at 65°C for 10 min. The phosphorylated oligonucleotides and the single-stranded antisense template of the rat PTH/PTHrP receptor cDNA, carried on the pcDNA1 plasmid vector (Invitrogen, San Diego, CA), were heated to 70°C for 2 min and then allowed to anneal by slowly cooling at room temperature.
Complementary DNA was synthesized by sequentially incubating the mixture on ice for 5 min, at room temperature for 5 min, and then at 37°C for 90 min in the presence of single-stranded binding protein, T4 ligase (2.5 units), and unmodified T7 DNA polymerase (1 unit), in a buffer (pH 7.4) containing Tris (17.4 mM), ATP (0.75 mM), dNTP (0.4 mM, each), MgCl 2 (0.4 mM), and dithiothreitol (21.5 mM). The reaction was terminated by adding 10 l of "stop" buffer (pH 8.0), containing Tris (10 mM) and EDTA (10 M). The synthesized cDNA was then introduced into Escherichia coli strain MC1061/p3 by electroporation (25 microfarads/2.5 kV/400 ohm). The sequence of the each mutant cDNA was verified by sequence analysis.
Transient Transfection of COS-7 and HEK293 Cells-All plasmid DNAs were prepared by the CsCl method (24) and stored at Ϫ20°C in sterile buffer (pH 7.5) containing Tris (10 mM), EDTA (1 mM) at a concentration of 1 g/l, as established by absorbance at 260 nm. Their quality and quantity also were assessed by agarose gel electrophoresis. Before transfection, a given amount of the plasmid DNA was diluted in 190 l of phosphate-buffered saline (PBS, pH 7.42), and this mixture was further diluted in 760 l of a DEAE/dextran (10 mg/ml) solution. The empty pcDNA1 plasmid or pcDNA1 containing the WT or mutant DNAs was introduced into COS-7 or HEK293 cells by a slight modification of the DEAE/dextran transfection method (20). In brief, cells in 150-mm dishes were gently washed (2 ϫ) with pre-warmed PBS and exposed to DMEM medium (19 ml) containing 10% Nuserum (Life Technologies, Inc.) and chloroquine (100 M). The plasmids in the DEAE-dextran solution were added to the cells for 3 h at 37°C; the transfection medium was aspirated, and the cells then were shocked by exposure to PBS containing 10% dimethyl sulfoxide at room temperature for 2 min. The cells were cultured in fresh DMEM medium supplemented with 7% FBS. Twenty-four h later, they were replated into 24-or 6-multi-well plates at a density of 50 -100,000 cells/cm 2 . In some experiments, cells in multi-well plates were directly transfected under the same condition with comparable concentrations of cDNA and transfection medium. Results from the two methods were indistinguishable. Cells were studied 60 -72 h after transfection.
Stable Transfection of LLC-PK1 Cells-WT and mutant PTH/PTHrP receptor constructs were introduced into LLC-PK1 cells, together with a neomycin gene, by the calcium phosphate method (22,25), and clones expressing these receptors were selected by growth in G418 (Life Technologies, Inc.). In brief, cells in 100-mm dish were rinsed (2 ϫ) with warm PBS and preincubated at 37°C for 30 -60 min in DMEM (5 ml), containing Hepes (pH 7.12, 20 mM) and 7% FBS. Calcium phosphate precipitates (500 l) of the mixture containing receptor cDNAs and pSV2neo plasmid DNA, in Hepes buffered salt solution, were added to the dish and further incubated at 37°C for an additional 6 h. Cells were shocked at 22°C for 3 min with 15% glycerol/Hepes buffered salt solution and washed gently (2 ϫ) with warm PBS, and the medium then was renewed. Eighteen to 20 h later, cells were replated into two 150-mm dishes in DMEM containing 7% FBS and 1 mg/ml G418. Selected colonies were propagated, and cells were further characterized for their capacity to bind 125 I-PTHrP and a receptor-specific polyclonal antibody, G48 (see below), and for their signal transduction properties. Cells were routinely stored in liquid nitrogen.
Radioreceptor Assay and Scatchard Analysis-Transfected cells, grown in 24-well plates, were washed (ϫ 2) with binding buffer (pH 7.7), which contained Tris (50 mM), NaCl (100 mM), KCl (5 mM), CaCl 2 (2 mM), 5% heat-inactivated horse serum, and 0.5% FBS. For radioreceptor assays, cells were incubated in binding buffer with 125 I-[Tyr 36 ]PTHrP-(1-36)-NH 2 (PTHrP, 100,000 cpm/500 l/well), in the absence or presence of unlabeled PTHrP (10 Ϫ11 to 10 Ϫ6 M) at 15°C for 4 h. These incubation conditions and conditions for preparing the radioiodinated ligand have been described (20). Cells then were washed (3 ϫ) with ice-cold binding buffer and solubilized in 0.5 ml of 0.1% SDS solution. Radioactivity in the cell lysate was measured by ␥-counting (Micromedic System Inc., Model 6/400 plus). Scatchard analysis was performed as described previously (20). Protein concentration was determined (Bio-Rad) in an aliquot of the lysate using bovine serum albumin as the standard. Cells per well were quantified using a hemocytometer, after cells in two wells from each plate had been suspended by trypsinization.
Quantification of PTH/PTHrP Receptors Expressed on the Cell Surface-Cells grown in 24-well plates were washed (3 ϫ) with PBS (pH 7.4), containing 5% FBS (FBS/PBS), and then incubated at room temperature for 2 h in FBS/PBS in the presence of sheep receptor-specific antibody G48 (1:500), or preimmune sheep serum (1:500) (20). G48 was developed against a synthetic 18-amino acid peptide (DKGWTPASTS-GKPRKEKA) of the rat PTH/PTHrP receptor's extracellular N-terminal region, and it was purified by affinity chromatography with antigen immobilized to Sepharose 4B by the CNBr method (26). Cells were then washed thoroughly (4 ϫ) with 5% FBS/PBS, incubated first for 1 h at room temperature with affinity-purified rabbit anti-sheep IgG (H ϩ L) (Kirkland & Perry Laboratories, Inc., Gaithersburg, MD), and then for an additional hour with 100,000 cpm/250 l/well of goat anti-rabbit 125 I-IgG (DuPont NEN). The incubation was terminated by washing the cells with PBS (3 ϫ); the cells were solubilized in 1 N NaOH, and the radioactivity was counted.
Measurement of Intracellular cAMP Accumulation-Cells grown in 24-well plates were incubated in serum-free DMEM medium, containing Hepes (pH 7.42, 10 mM) with 3-isobutyl-1-methylxanthine (1 mM) and 0.1% bovine serum albumin at room temperature for 10 min. Then, COS-7 and LLC-PK1 cells were incubated at 37°C in the absence or presence of rPTH (10 Ϫ12 to 10 Ϫ6 M) for an additional 15 and 5 min, respectively. The reaction was terminated by aspirating the medium, washing the cells with ice-cold PBS (2 ϫ), and freezing the cells in 500 l of HCl (50 mM). The HCl extracts were stored at Ϫ80°C until intracellular cAMP was measured by radioimmunoassay, as described previously (20). Cyclic AMP accumulation (pmol/well) is expressed as stimulated minus basal values.
Measurement of Accumulated Inositol Phosphates-Accumulated IPs were measured in cells grown in 6-well plates by methods described previously (20,22). Briefly, cells were prelabeled at 37°C for 8 -12 h with 3 Ci/ml [ 3 H]myoinositol in inositol-and serum-free DMEM (Life Technologies, Inc.), supplemented with 0.1% bovine serum albumin. Cells then were washed (2 ϫ) with prewarmed inositol-and serum-free DMEM containing LiCl (30 mM) and treated with rPTH (10 Ϫ11 to 10 Ϫ6 M) or with vehicle alone (10 mM acetic acid) at 37°C for an additional 30 min. The reactions were terminated by aspirating the incubation medium and adding 1 ml of ice cold 5% trichloroacetic acid. Trichloroacetic acid extractions and separation of the IPs by AG 1X8 anion exchange column chromatography (formate form, 100 -200 mesh, Bio-Rad) were performed as described previously in detail (20). Specific PTH-stimulated phosphoinositide hydrolysis was determined by subtracting basal values from the total values obtained by PTH stimulation. Radioactivity in each fraction was counted by liquid scintillation counting (Beckman, Model LS 6000IC).
Measurement of Sodium-dependent Phosphate Co-transport in LLC-PK1 Cells-LLC-PK1 cells stably expressing WT or mutant PTH/ PTHrP receptors were grown in 24 multi-well plates, and subsequently they were incubated with rPTH-(1-34) (10 Ϫ10 to 10 Ϫ6 M) at 37°C for 6 h, prior to measurements of sodium-dependent phosphate co-transport, as described previously in detail (22,25). Briefly, cells were washed (2 ϫ) with warm phosphate-free Hepes buffer (pH 7.42, 10 mM) that contained NaCl (150 mM), KCl (5 mM), MgCl 2 (1.8 mM), CaCl 2 (1.0 mM), and glucose (5 mM) and then further incubated in the same buffer which now contained 0.2 mM phosphate (pH 7.42) and 0.5 Ci/250 l/well [ 32 P]orthophosphate at 37°C for 5 min. The reaction was terminated by placing the plate on ice and washing the cells (3 ϫ) with ice-cold Hepes stop buffer (pH 7.42, 10 mM), which contained choline chloride (150 mM) and sodium arsenate (5 mM). Cells then were solubilized in NaOH (1 N), and the radioactivity in the lysates was measured by liquid scintillation counting. Protein concentration was measured in an aliquot of each lysate. The initial velocity of phosphate uptake was expressed as nmol/5 min/mg protein.
Statistical Analysis-Means Ϯ S.D. of the three to six replicates were calculated from each of two or more independent experiments. One-way analysis of variance followed by Student's t test were used to determine significance (p Ͻ 0.05).

Dual Signaling by the PTH/PTHrP Receptor through Adenylyl Cyclase and PLC Pathways Is Dissociated by Mutations in Its 2i
Loop-The capacity of the rat WT PTH/PTHrP receptor, with SEKKY at positions 316 -320 in the 2i loop, to increase intracellular accumulation of cAMP and IPs was compared with those of four receptors with random clustered mutations at the same site (SDSEL, GRELG, RTKKS, and SKEKY) in COS-7 cells (Fig. 1). Expression of all receptors also was measured by ligand binding and by binding of the receptor-specific antiserum, G48. PTH (100 nM)-stimulated accumulation of IPs was severely impaired in cells expressing receptors with the DSEL clustered mutation, whereas cAMP accumulation was indistinguishable from that of cells expressing WT receptors (Fig. 1). Cell-surface expression of this mutant was only slightly impaired, if at all, and Scatchard analysis showed that the binding affinities of WT and DSEL mutants were indistinguishable, with apparent K d values of 2.3 Ϯ 0.3 and 2.5 Ϯ 0.4 nM, respectively (data not shown). Results from cells expressing receptors with the three other clustered mutations were less informative. Receptors with either RTKKS or SKEKY clustered mutations showed no significant impairment in their signal transduction properties. PTH (100 nM)-generated IPs in cells expressing receptors with the GRELG mutation was not totally abolished, although it was markedly impaired, compared with the WT receptor (20 Ϯ 8%, n ϭ 4). We thus focused attention on receptors with the DSEL mutant cassette.
Since we had previously shown that PTH-stimulated accumulation of IPs was strikingly dependent on the density of receptors expressed on the cell surface (20,22), we next examined the relationship between PTH-stimulated accumulation of IPs and cAMP, with expression of WT and DSEL mutant receptors. Receptor expression was proportional to the amount of cDNA used for transfection, up to about 10 g/10-cm dish, and expression of PTH/PTHrP receptors with the DSEL mutant cassette was only slightly less efficient than that of WT receptors ( Fig. 2A). PTH (100 nM)-generated IPs increased with increased expression of WT PTH/PTHrP receptors, whereas no detectable accumulation of IPs was observed in cells transfected with the DSEL mutant receptor at any expression level (Fig. 2B). In contrast, PTH (100 nM)-generated cAMP increased with increased expression of either WT or mutant receptor (Fig. 2C).
Point Mutations in the Rat PTH/PTHrP Receptor 2i Loop Diminish, but Do Not Abolish PTH-stimulated PLC Activity-To define more precisely the amino acids essential for PTH-generated IPs, we constructed mutant receptors in which each amino acid of the WT sequence, EKKY, from residues 317 to 320 in the 2i loop was replaced by alanine or the corresponding amino acid in the DSEL sequence. Glu-317, Lys-318, and Lys-319 were also replaced by other charged amino acids, and Tyr-320 was replaced with Phe. All these mutant receptors were relatively well expressed; G48 binding was 79 -107% and maximal radioligand binding (B 0 ) was 92-112%, respectively, compared with the WT receptor (Table I). Although maximal PTH-generated cAMP was minimally altered by mutations in these four amino acids, accumulation of IPs differed strikingly (Table I and Fig. 3). The signal transduction properties of the PTH/PTHrP receptor were most influenced by modifications at Lys-319. K319E selectively decreased the receptor's capacity to generate IPs to 27 Ϯ 13%, compared with the WT receptor, but it did not eliminate it. In contrast, K319A modestly reduced the receptor's capacity to signal through both signal transduction pathways. Interestingly, the signaling properties of a mutant receptor in which K319R were indistinguishable from those of the WT receptor. Modifications of Lys-318 modestly changed the receptor's capacity to transduce signals, and these changes were nonselective: K318S reduced signaling through both adenylyl cyclase and PLC pathways; K318E increased signaling through both pathways; and K318A did not affect receptor signaling. Furthermore, the phenotype of a double mutant receptor in which Lys-318 and Lys-319 both were Glu closely resembled that of K319E, rather than K318E, thus reflecting the greater importance of the amino acid at position 319 (Table  I, Fig. 3).
Mutating E317A did not affect PTH-stimulated accumulation of IPs, but PTH-generated IPs increased slightly in cells expressing receptors with E317D or E317K. None of these mutations at position 317 affected PTH-generated cAMP accumulation. Changing Tyr-320 to Ala, Phe, or Leu did not affect receptor signaling (Table I, Fig. 3).

PTH-stimulated PLC Activity in Rat PTH/PTHrP Receptors with Mutations in the 2i Loop Is Restored by a Single-point Revertant in Which the Glutamic Acid at Position 319 Is
Replaced with Lysine, the WT Amino Acid-We then modified the rat PTH/PTHrP receptor with the DSEL mutant cassette by individually introducing the WT amino acids back at their original positions in the sequence and assessing their functional properties. Replacing the mutant E319K (DSKL) fully restored its capacity to increase accumulation of IPs (Fig. 4, Table I). Furthermore, PTH-generated cAMP and IPs in cells expressing this revertant had dose-response characteristics that were indistinguishable from the WT receptor: the EC 50 values of the WT and this mutant receptor, respectively, for accumulation of IPs were 25 Ϯ 3 and 23 Ϯ 4 nM (Fig. 5A) and for cAMP accumulation were 0.32 Ϯ 0.04 and 0.37 Ϯ 0.05 nM (Fig. 5B). PTH treatment of cells expressing the receptor with the DSEL mutant cassette failed to generate IPs even at doses as high as 1 M (Fig. 5A), whereas cAMP responsiveness to incremental doses of PTH was indistinguishable from that of the WT receptor.
On the other hand, replacing the other amino acids in the DSEL mutant cassette with the corresponding WT residue partially restored the receptor's capacity to generate IPs; PTHgenerated IPs in cells expressing receptors with D317E (ESEL), S318K (DKEL), and L320Y (DSEY) were 26 Ϯ 15, 22 Ϯ 9, and 12 Ϯ 4%, respectively, compared with the WT receptor (Fig. 4, Table I). These data thus confirm the importance of Lys-319 for signaling PLC, but they also suggest a minor role for neighboring amino acids in the 317-320 sequence.

Co-expression of G␣ q and with Either the WT Receptor or a Mutant PTH/PTHrP Receptor with the DSEL Mutant
Cassette-To establish whether the mutant PTH/PTHrP receptor with the DSEL mutant cassette was incapable of generating IPs because the levels of G␣ q were inadequate, we compared the properties of the WT with this mutant receptor in cells co-transfected with G␣ q . PTH treatment of cells that co-expressed the DSEL mutant receptor together with G␣ q did not significantly increase IPs in either HEK293 cells or LLC-PK1 cells. In contrast, PTH-stimulated accumulation of IPs in both HEK293 and LLC-PK1 cells was enhanced by 2-and 2.5-fold, respectively, in the cells expressing WT PTH/PTHrP receptor and G␣ q , compared with PTH-treated cells expressing only the WT rat PTH/PTHrP receptor (Fig. 6). Overexpression of G␣ q did not increase basal PLC activity in either cell. Thus, G␣ q overexpression did not overcome the signaling defect of PTH/ PTHrP receptors with the DSEL mutant cassette.

A PLC-dependent Biological Function in LLC-PK1 Cells Is Lost in Cells Stably Expressing PTH/PTHrP Receptors with the DSEL Mutant
Cassette-We previously reported that PTH augments sodium-dependent phosphate co-transport in LLC-PK1 cells that stably express the rat WT PTH/PTHrP receptor and further that this response appeared to depend on activation of PLC and protein kinase C (22,25). To determine whether mutant PTH/PTHrP receptors with the DSEL mutant cassette would exhibit the same properties in LLC-PK1 cells as they did in COS-7 cells and whether sodium-dependent phosphate cotransport was linked to the PLC response, we isolated LLC-PK1 subclones following stable transfection with the WT receptors or receptors with the DSEL mutant cassette. As we previously had shown that PLC stimulation and phosphate transport were strongly dependent on the level of surface receptors expressed (20, 22), we compared the function of the WT and the mutant receptor in subclones that expressed comparable numbers of receptors (approximately 200,000 sites per cell). PTH increased both IP 3 accumulation and phosphate uptake, dose-dependently, over a similar range of PTH concentrations (0.1 to between 100 and 1000 nM) only in cells expressing the WT receptor. Even at PTH doses as high as 1 M, however, neither intracellular IP 3 accumulation nor phosphate uptake was affected in cells expressing receptors with the DSEL mutant cassette (Fig. 7). PTH-stimulated cAMP accumulation was indistinguishable, however, in cells expressing either WT receptors or receptors with the DSEL mutant cassette (data not shown).

DISCUSSION
These studies provide a clear demonstration that the capacity of the PTH/PTHrP receptor to signal through the PLC pathway can be selectively abolished, without affecting its capacity to activate adenylyl cyclase. Furthermore, they define some of the structural requirements necessary for signaling by this receptor through members of the G q family. Preliminary analysis revealed that cells expressing the rat PTH/PTHrP receptor, in which four sequential amino acids, EKKY (amino acids 317-320) in its 2i loop were mutated to DSEL, totally lacked the capacity to generate IPs when treated with PTH, although the mutant receptor was well expressed, and its PTHstimulated cAMP response was indistinguishable from the WT receptor.
Extensive investigation of the role of each amino acid in this region pointed to residue 319 as the site whose sole modification caused the greatest reduction in PLC signaling. Replacing Lys-319 with Glu maximally decreased PTH-generated IPs to 27 Ϯ 13%, compared with the response of the WT receptor, but,  3. Maximal PTH-generated IPs and cAMP in COS-7 cells expressing receptors with the DSEL mutant cassette or with single-or double-point mutations in the 2i loop are compared with rat WT PTH/PTHrP receptor. COS-7 cells were transfected with the respective plasmid DNAs, and their capacity to generate IPs and cAMP in response to rPTH (100 nM) was measured, as described under "Experimental Procedures." The properties of the mutant receptors are compared with those of the WT receptor, which has been set as 100%. Accumulated IPs and cAMP in cells expressing the mutant receptors have been adjusted to reflect their level of expression, using the formula: corrected PTH-stimulated second messenger concentration in the mutant receptor equals PTH-stimulated second messenger concentration in the mutant receptor ϫ (G48 binding to WT receptor/G48 binding to the mutant receptor) ϫ 100, where the properties of the WT receptor have been set at 100%. For uncorrected data and the number of replicate experiments for each construct see Table I.  Table I. importantly, no single substitution completely abolished signaling through the PLC pathway. It seems likely, in retrospect, that PTH-generated IPs was markedly reduced in receptors with the clustered mutation, GRELG at position 316 -320, because of K319L mutation. The importance of Lys-319 for generating IPs was further substantiated by showing that a revertant in which Glu-319 in the DSEL mutant cassette was replaced with WT amino acid, Lys, restored full activation of PLC by PTH. However, PTH/PTHrP receptor-G q interactions are more complex, as evidenced by our finding that revertants in which individual WT amino acids replaced the mutant residues at positions 317, 318, and 320 in the DSEL mutant cassette (D317E, S318K, L320Y) also partially restored PTHgenerated IPs. The contributions of any individual amino acid for activating the PLC pathway, therefore, must be considered within the context of elaborate interactions between PTH/ PTHrP receptors and G q heterotrimers.
The rat WT PTH/PTHrP receptor's PLC-stimulating capacity was indistinguishable from that of receptors with K319R. It was also modestly reduced (64 Ϯ 11% of WT), however, when Lys-319 was replaced with the neutral amino acid, Ala. These data, together with experiments in which full PLC activation was restored by changing Glu-319 in the DSEL sequence back to Lys, the WT amino acid, suggest that a positively charged amino acid at position 319 may be important, although not absolutely necessary, for activating the relevant member(s) of the G q family.
The lysine at position 318 in the WT rat PTH/PTHrP receptor appeared to be of only minor importance for coupling to G q proteins. Replacing Lys-318 with Ala, Ser, or Glu affected the receptor's phenotype only modestly, and these changes were nonselective, that is they simultaneously decreased or increased accumulation of both IPs and cAMP. Support for the notion that Lys-319 is more important than Lys-318 for signaling through the PLC pathway also comes from examining the properties of the PTH/PTHrP receptor with both K318E/K319E mutations. The capacity of mutant receptor to generate second messengers closely resembled that of receptors with K319E, rather than K318E. Furthermore, these data demonstrate that in contrast to position 319, a positively charged amino acid at position 318 is not critical for receptor function.
The properties of receptors with single-point mutations, in which Glu-317 was replaced with Ala, Asp, or Lys, or Tyr-320 was replaced with Phe, Ala, or Leu, were closely similar to or indistinguishable from those of the WT receptor. These data indicate that the amino acids at these positions probably are not integrally involved in coupling to G q .
Our data are consistent with the notion that receptors containing the DSEL mutant cassette might couple to G q proteins but that this interaction is only less efficient. If this were the case, overexpression of G␣ q , together with this mutant receptor, might overcome the signaling defect in this mutant receptor. However, PTH failed to generate IPs when receptors with the DSEL mutant cassette were expressed together with G␣ q in either HEK293 or LLC-PK1 cells. This sharply contrasted with the 2-and 2.5-fold increase in the PLC response in HEK293 and LLC-PK1 cells that co-expressed both the rat WT PTH/ PTHrP receptor and G␣ q . Since a PTH/PTHrP receptor with the DSEL mutant cassette had unimpaired cAMP responsiveness and thus must release ␤␥-subunits from the activated heterotrimeric Gs, these data strengthen the argument that stimulation of PLC by PTH in cells expressing the WT receptor is mediated by G␣ subunit(s), rather than via activation of ␤␥, as has been reported with some G protein-coupled receptors (27)(28)(29)(30). Presumably, the DSEL mutant cassette in the PTH/ PTHrP receptors' 2i loop strongly impairs interaction with G␣ q . Furthermore, our findings in cells co-expressing the WT PTH/ PTHrP receptor and G␣ q extend our earlier observations that co-expression of any one of four G␣ q proteins, G␣ q , G␣ 11 , G␣ 14 , and G␣ 16 , together with the WT rat PTH/PTHrP receptor, markedly increased PTH-stimulated accumulation of IPs (31) and that the PTH-stimulated sodium-dependent phosphate cotransport in LLC-PK1 cells stably expressing the rat PTH/ PTHrP receptor is cAMP-independent and apparently mediated through PLC (25).
Treating all members of the PTH/PTHrP/secretin/calcitonin receptor family with their cognate agonists generates cAMP. In many, and perhaps all, of these receptors agonist occupancy also activates the PLC pathway. For example, activated receptors for pituitary adenylyl cyclase-activity peptide (PACAP) (32), glucagon (33), and calcitonin (34,35) increased both intracellular IPs and free calcium. Also, activated corticotropinreleasing hormone (CRF) receptors generated IPs (36), and activated receptors for both gastric inihibitory peptide (GIP) and glucagon-like peptide 1 (GLP-1) increased intracellular free [Ca 2ϩ ] (33,37,38). Aligning the amino acids of members of this receptor family reveals that the EKKY sequence is identical in all five mammalian PTH/PTHrP receptors, and it is highly homologous with the sixth from Xenopus laevis, where the only change is Asp, rather than Glu, at the corresponding position (39). Three of these amino acids, Asp, Lys, and Tyr, also are conserved in the PTH2 receptor sequence, including the Lys corresponding to Lys-319 in the rat PTH/PTHrP receptor (3). Among the other members of this receptor family, EKKY in the PTH/PTHrP receptor aligns with ERKY in the vasoactive intestinal peptide (VIP) and secretin receptors and with ERRY in the PACAP receptor; Lys-318 and Lys-319 align with positively charged amino acids, RR, in the growth hormone-releasing peptide (GHRH) receptor. Homology is less evident, however, with other members; EQR in the glucagon receptor correspond with EKK in the PTH/PTHrP receptor, and ER in the GIP receptor are positioned identically with Glu-317 and Lys-318 in the PTH/PTHrP receptor, although Ser aligns with Lys-319 in the PTH/PTHrP receptor. There is no apparent sequence homology among calcitonin, GLP-1, and PTH/PTHrP receptors in this portion of the 2i loop, except for Glu, which corresponds to Glu-317 (Fig. 8). The residues important for PLC signaling by these other receptors have not yet been defined.
All G protein-coupled receptors are thought to have seven membrane-spanning helices. Many of them couple to G q (s), including adrenergic, muscarinic, and glycoprotein hormone receptors. Activation of glycoprotein hormone receptors, like activation of PTH/PTHrP receptors, also stimulates both adenylyl cyclase and PLC (40 -42). Therefore, relevant features that enable them to couple to G q possibly might be gleaned from examining their primary structure, despite their lack of amino acid homology with PTH/PTHrP receptors. Few paradigms have emerged, however, concerning determinants important for coupling these receptors to G q proteins. All three intracellular loops, especially 2i and 3i, as well as the Nterminal portion of the cytoplasmic tail have been implicated in PLC activation by adrenergic, muscarinic, and glycoprotein hormone receptors, although the putative interactive sites vary among the individual receptors (43)(44)(45)(46)(47). There are several examples where agonist-stimulated PLC activity of a G proteincoupled receptor is severely affected, when a single amino acid is mutated. The TSH receptor's capacity to generate IPs, for example, was markedly impaired when alanine at position 623 in the 3i loop was mutated to lysine or glutamic acid, but its capacity to activate adenylyl cyclase was unimpaired (48). It is puzzling, however, that the ␣1B-adrenergic receptor became constitutively activated with respect to stimulating PLC, when alanine at position 293, which is located at the same relative position as alanine 623 in the TSH receptor, was replaced with any amino acid (49). Mutating a highly conserved leucine, putatively located in the middle of the 2i loop of the GnRH, m1, and m3 muscarinic receptors to alanine or to a polar amino acid also markedly reduced agonist-generated IPs, although like mutations of Lys-319 in the PTH/PTHrP receptor, PLC activity was not totally eliminated (50 -52). Moreover, based on evidence that a Phe to Ala mutation at the same relative position in the ␤ 2 -adrenergic receptor markedly reduced isoproterenolgenerated cAMP, these authors concluded that the a bulky hydrophobic residue at this site played a nonspecific role in coupling G protein-coupled receptors to more than one G protein (50). In recent studies of mutant ␣1B-adrenergic receptors, in which charged residues in the highly conserved 2i loop sequence, DRY, were substituted, Scheer et al. (53) showed that D142A conferred high constitutive activity, whereas R143A or Asn markedly reduced agonist-stimulated accumulation of inositol phosphate. These findings were consistent with predictions from molecular dynamic simulations and led the authors to suggest that the main role of Arg-143 is to mediate receptor activation, by allowing several amino acids in the 2i and 3i loops to attain a configuration that facilitates docking with the G protein.
Our results establish that polyphosphoinositol production by the activated PTH/PTHrP receptor can be selectively abolished by mutations in the 2i loop, without affecting its capacity to generate cAMP, and that Lys-319, or perhaps any basically charged amino acid at this position, is important for coupling the PTH/PTHrP receptor to the relevant G q (s). Moreover, activation of the PLC pathway by the PTH/PTHrP receptor is mediated by ␣-subunits of the G q family, rather than by ␤␥-subunits, and sodium-dependent phosphate co-transport in LLC-PK1 cells is mediated through the PLC, rather than the adenylyl cyclase, pathway. Future characterizations of receptor mutants together with structural analyses will ultimately be necessary to define these complex receptor-G protein interactions.