L-phenylalanine and NPS R-467 synergistically potentiate the function of the extracellular calcium-sensing receptor through distinct sites.

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) can be potentiated by allosteric activators including calcimimetics and l-amino acids. In this study, we found that many mutations had differential effects on the functional modulation of the CaR by these two allosteric activators, supporting the idea that these modulators act through distinct sites. 10 mm l-phenylalanine and 1 microm NPS R-467, submaximal doses of the two agents, each elicited similar modulation of R185Q. However, there are different relative potencies for these two modulators with some receptors being more responsive to l-phenylalanine and others being more responsive to NPS R-467. The responsiveness of the CaR to Ca(2+)(o) appears to be essential to observe the potentiating action of l-phenylalanine but not of NPS R-467 on the receptor. NPS R-467 reduces the Hill coefficients of the wild-type as well as mutant receptors, suggesting that engagement of all Ca(2+) binding sites is not required when the receptor is activated by NPS R-467. In contrast, l-phenylalanine has little effect on the Hill coefficients of mutant receptors. The two-site model is further supported by the observation that these two classes of modulators exert a synergistic effect on CaRs with inactivating mutations that are responsive to both modulators.

The extracellular calcium (Ca 2ϩ o )-sensing receptor (CaR) 1 plays a key role in mineral ion homeostasis by sensing small perturbations in the level of Ca 2ϩ o and modulating the functions of the parathyroid and the kidney so as to restore Ca 2ϩ o to its normal level (1). The CaR has been identified as an important therapeutic target for disorders of systemic Ca 2ϩ o homeostasis such as primary and secondary hyperparathyroidism. Earlier studies have documented that the CaR can be activated allosterically by L-amino acids and the phenylalkylamine calcimimetics in the presence of Ca 2ϩ o at millimolar levels (2). In turn, these allosteric modulators stereoselectively enhance the sensitivity of the CaR to its polycationic agonists such as Ca 2ϩ o and spermine. It has been documented that calcimimetics act through the transmembrane domain of the receptor (3). The CaR is a G protein-coupled receptor in the same subfamily C as the metabotropic glutamate receptors (mGluRs) 1 through 8 (4 -6). Five of the residues in the glutamate binding site of mGluR1a in the amino-terminal extracellular domain are conserved in the CaR. According to Zhang et al. (9), mutations at these sites significantly attenuate the responsiveness of the receptor to its principal physiological agonist, Ca 2ϩ o , suggesting possible homology between the glutamate binding site of the mGluRs and the calcium binding site of the CaR. Ser-170 whose conserved residue, Thr-188, in the mGluRs is involved in glutamate binding is also important for the modulation of the CaR by L-phenylalanine. Alanine substitution of Ser-170 along with substitutions of two other serines at positions 169 and 171 was found to completely block the response of the CaR to L-phenylalanine. Therefore, L-phenylalanine appears to act through a site in the extracellular domain of the CaR to enhance receptor function.
In the present studies, we examined the effects of a calcimimetic, NPS R-467, on several mutant receptors and compared the results with the effects of L-phenylalanine or both agents together on the same CaRs to functionally demonstrate that these two types of allosteric modulators act on the CaR through distinct sites.

Transient Expression of CaRs in Human Embryonic Kidney (HEK)
293 Cells-The DNA for transfection was prepared using the plasmid Midi Kit (Qiagen) (7). LipofectAMINE (Invitrogen) was employed as a DNA carrier for transfection. The HEK293 cells used for transient transfection were provided by NPS Pharmaceuticals (Salt Lake City, UT) and cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Hyclone). The DNA-liposome complex was prepared by mixing DNA and LipofectAMINE in Opti-MEM I reduced serum medium (Invitrogen) and incubating the mixture at room temperature for 30 min. The DNA-LipofectAMINE mixture was then diluted with Opti-MEM I reduced serum medium and added to 90% confluent HEK293 cells plated on 13.5 ϫ 20.1-mm glass coverslips using 2.5 g of DNA. After a 5-h incubation at 37°C, equivalent amounts of Opti-MEM I reduced serum medium with 20% fetal bovine serum were added to the medium overlying the transfected cells, and the medium was replaced with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 24 h after transfection. The expressed CaR protein was assayed 48 h after the start of transfection. To co-express two receptors, 1.25 g of each of the two cDNAs was mixed and used to transfect HEK293 cells.
Measurement of Ca 2ϩ i by Fluorimetry in Cell Populations-Coverslips with HEK293 cells previously transfected with the appropriate CaR cDNAs were loaded for 2 h at room temperature with Fura-2/AM in 20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , 0.1% bovine serum albumin, and 0.1% dextrose and then washed once with a bath solution (20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% dextrose, and 0.1% bovine serum albumin) at 37°C for 20 min. The coverslips were then placed diagonally in a thermostated quartz cuvette containing the bath solution using a modification of the technique employed previously in this laboratory (7). The L-phenylalanine and/or NPS R-467 were added into the bath solution and preincubated for 50 s. Concentrated Ca 2ϩ o at 100 mM or 1 M was added stepwise to an experimental solution containing 0.5 mM Ca 2ϩ to achieve the following concentrations: 1.5, 2.5, 3.5, 4.5, 5.5, 10, 15, 20, 30, 40, and 50 mM. Excitation monochrometers were centered at 340 and 380 nm, and emission light was collected at 510 Ϯ 40 nm through a wide band emission filter. The 340/380 excitation ratio of emitted light was used to estimate Ca 2ϩ i as described previously (7). o was performed using ANOVA or Duncan's multiple comparison test (p Յ 0.05) (8). Each of the experiments presented in the results was performed at least three times. To calculate the EC 50 , the maximal response, and the Hill coefficient, we used the MacFitCurve program and the Hill equation F(X) ϭ a⅐Xĥ /(eĥ ϩ Xĥ ) where "F " is a variant as a function of X, "F(X)" is the response at a given concentration with agonist (X), "a" is maximal response, "e" is EC 50 , and "h" is Hill coefficient to fit the experimental data.

RESULTS
We compared the modulatory effects of a phenylalkylamine calcimimetic and an L-amino acid on the wild-type and various mutant CaRs. Our initial studies examined the effects of 1 M NPS R-467 and 10 mM phenylalanine on the wild-type receptor. NPS R-467 decreased the EC 50 value of the wild-type CaR by 39%, whereas L-phenylalanine decreased it by 16% ( Fig. 1A and Table 1). However, NPS R-467 markedly decreased the maximal responses elicited by high concentrations of Ca 2ϩ o (Table I). Thus, the extent of receptor activation in the presence of 1 M NPS R-467 or 10 mM L-phenylalanine was similar at physiological concentrations of Ca 2ϩ o . Unlike phenylalanine, NPS R-467 also significantly reduced the Hill coefficient of the wild-type receptor ( Table I), suggesting that the requirement for Ca 2ϩ obinding is different for phenylalanine and NPS R-467.
Similar to phenylalanine as demonstrated by Zhang et al. (9), NPS R-467 significantly increased the Ca 2ϩ o -elicited maximal responses of several mutant receptors. For instance, NPS R-467 and L-phenylalanine each increased the maximal response of R185Q to a similar extent ϳ1.4-fold (Fig. 1B). NPS R-467 and L-phenylalanine also substantially decreased the EC 50 value of R185Q to a greater extent than that seen for the wild-type CaR. There appears to be a limit to the positive modulation of the wild-type CaR that can be elicited by an addition of the calcimimetic or L-phenylalanine. Mutant receptors with less sensitivity to polycationic agonists and reduced maximal activation often showed greater potentiation by the two modulators than the wild-type receptor. The effects of NPS R-467 or L-phenylalanine were not always identical when other mutant receptors were studied as described below.
Many mutant receptors with substitutions of conserved amino acid residues within the putative amino acid binding site in the extracellular domain remained responsive to L-phenylalanine. S170A and Y218S each responded to L-phenylalanine with significant increases in the magnitude of the response at all of the tested concentrations of Ca 2ϩ o . With NPS R-467, all of the responses of the two receptors were further increased to even greater levels (Fig. 2), and sensitivities of the respective receptors to Ca 2ϩ o were also enhanced as indicated by the significant declines in the EC 50 values (Table I). In contrast, S147A, D190A, and E297K showed greater increases in their responses to L-phenylalanine than to NPS R-467 (Fig. 3). In the presence of either L-phenylalanine or NPS R-467, the EC 50 values for these three mutant receptors were also significantly lowered (Table I). Therefore, the responsiveness of the mutant receptors to L-phenylalanine and NPS R-467 were not uniformly affected by mutations, strongly suggesting that these two modulators act through distinct binding sites.
The mutant receptors G143E, R795W, S169A/S170A/S171A showed weak or no activation by the physiological agonist Ca 2ϩ o and also showed no potentiation by L-phenylalanine. In contrast, NPS R-467 enhanced the responses of each of these mutant receptors to Ca 2ϩ o (Fig. 4). G143E is a special case where there was no activation by Ca 2ϩ o unless NPS R-467 was also present, indicating that the responsiveness of the CaR to agonist stimulation is not essential for its modulation by NPS R-467 and that these two allosteric activators act on the receptor through different mechanisms.
Heterodimeric receptors were also affected differently by i responses. Before the additions of Ca 2ϩ o , the cells were not exposed (circle), exposed to 1 M NPS R-467 (square), or exposed to 10 mM Phe (triangle). Responses are normalized to the maximal cumulative Ca 2ϩ i responses of the wild-type receptor in untreated cells. Each point is the mean value of the number of measurements indicated in Table I. Standard errors of the mean (ϮS.E.) are indicated with vertical bars through each point. Some error bars are smaller than the symbol. The data were fitted using MacCurveFit as described under "Experimental Procedures." these two types of modulators in some cases. For instance, heterodimers formed in cells co-transfected with G143E and the inactive mutant CaR, A877Stop, or in those cells co-transfected with S169A/S170A/S171A and A877Stop that recovered 60% of their Ca 2ϩ i responses to Ca 2ϩ o hardly responded to L-phenylalanine (Fig. 5). In contrast, NPS R-467 substantially reduced the EC 50 values of these heterodimers while eliciting modest increases in their maximal responses (Table I).
To further demonstrate that L-phenylalanine and NPS R-467 act on the receptor through different sites, we examined whether these two allosteric activators affected the function of the receptor synergistically when added together. The actions of both activators combined on the wild-type receptor resembled that of NPS R-467 alone (Fig. 6A). In contrast, the mutant receptor E297K, which has a very attenuated response in the absence of these two modulators, became significantly more active when these two modulators were combined than when they were applied individually at their maximal effective doses Triple is S169A/S170A/S171A with triple serines mutated to three alanines. FIG. 2. Allosteric modulation of mutant receptors S170A, Y218S, and S169A/S170A/S171A. HEK293 cells were transfected with S170A (A) or Y218A (B). Ca 2ϩ o -evoked Ca 2ϩ i responses were measured as described in Fig. 1. Before the additions of Ca 2ϩ o , the cells were not exposed (circle), exposed to 1 M NPS R-467 (square), or exposed to 10 mM Phe (triangle). Responses are normalized to the maximal cumulative Ca 2ϩ i responses of the wild-type receptor in untreated cells. Each point is the mean value of the number of measurements indicated in Table I. Standard errors of the mean (ϮS.E.) are indicated with vertical bars through each point. Some error bars are smaller than the symbol. The data were fitted using MacCurveFit as described under "Experimental Procedures." (Fig. 6B). The EC 50 was reduced by nearly half (18 -11 mM) in the presence of either 30 M NPS R-467 or 30 mM L-phenylalanine, whereas the presence of both agents resulted in a further reduction in the EC 50 to nearly 25% of its original level (18 -5 mM). As shown in Fig. 6, 30 M NPS R-467 and 30 mM Lphenylalanine were the maximal doses for these modulators; thus, doubling the concentration of either modulator had no further effect on receptor activity. Consistent with the lack of L-phenylalanine modulation of the mutant CaRs, G143E and S169A/S170A/S171A, synergistic effects were not observed FIG. 3. Allosteric modulation of mutant receptors S147A, D190A, and E297K. HEK293 cells were transfected with S147A (A), D190A (B), or E297K (C). Ca 2ϩ o -evoked Ca 2ϩ i responses were measured as described in Fig. 1. Before the additions of Ca 2ϩ o , the cells were not exposed (circle), exposed to 1 M NPS R-467 (square), or exposed to 10 mM Phe (triangle). Responses are normalized to the maximal cumulative Ca 2ϩ i responses of the wild-type receptor in untreated cells. Each point is the mean value of the number of measurements indicated in Table I. Standard errors of the mean (ϮS.E.) are indicated with vertical bars through each point. Some error bars are smaller than the symbol. The data were fitted using MacCurveFit as described under "Experimental Procedures. "   FIG. 4. Allosteric modulation of the mutant receptors G143E, R795W, and S169A/S170A/S171A. HEK293 cells were transfected with G143E (A), R795W (B), or S169A/S170A/S171A (C). Ca 2ϩ o -evoked Ca 2ϩ i responses were measured as described in Fig. 1. Before the additions of Ca 2ϩ o , the cells were not exposed (circle), exposed to 1 M NPS R-467 (square), or exposed to 10 mM Phe (triangle). Responses are normalized to the maximal cumulative Ca 2ϩ i responses of the wild-type receptor in untreated cells. Each point is the mean value of the number of measurements indicated in Table I. Standard errors of the mean (ϮS.E.) are indicated with vertical bars through each point. Some error bars are smaller than the symbol. The data were fitted using Mac-CurveFit as described under "Experimental Procedures." with the mutant receptors G143E (Fig. 6C) and S169A/S170A/ S171A (data not shown).

DISCUSSION
In this study, we compared the modulatory effects of NPS R-467 and L-phenylalanine, two allosteric activators of the CaR. Both compounds were found to potentiate the activation of the wild-type receptor as well as many mutant receptors with inactivating mutations. With the wild-type receptor, the primary effect observed with these modulators is a decrease in the EC 50 for activation of the CaR by Ca 2ϩ o . Most mutant receptors also showed a decrease in EC 50 . In addition, many of these receptors demonstrated increases in their maximal responses to agonist activation, which was not found in the wildtype CaR. Interestingly, NPS R-467 consistently decreased the EC 50 for the mutant receptors, whereas L-phenylalanine often showed little or no effect on EC 50 despite producing significant Allosteric modulation of heterodimeric receptors formed in cells co-transfected with G143E and A877Stop or S169A/S170A/S171A and A877Stop. In HEK293 cells, G143E (A) or S169A/S170A/S171A (B) was co-transfected with A877Stop. Ca 2ϩ oevoked Ca 2ϩ i responses were measured as described in Fig. 1. Before the additions of Ca 2ϩ o , the cells were not exposed (circle), exposed to 1 M NPS R-467 (square), or exposed to 10 mM Phe (triangle). Responses are normalized to the maximal cumulative Ca 2ϩ i responses of the wildtype receptor in untreated cells. Each point is the mean value of the number of measurements indicated in Table I. Standard errors of the mean (ϮS.E.) are indicated with vertical bars through each point. Some error bars are smaller than the symbol. The data were fitted using MacCurveFit as described under "Experimental Procedures." increases in the maximal responses of many mutant receptors. Another difference between NPS R-467 and L-phenylalanine is that NPS R-467 significantly decreased the maximal response of the wild-type receptor, whereas L-phenylalanine had no effect on this parameter in the wild-type receptor. The presence of the physiological polycationic CaR agonist Ca 2ϩ o is essential for the actions of both NPS R-467 and L-phenylalanine on the wild-type and mutant receptors.
We chose 1 M NPS R-467 and 10 mM L-phenylalanine as our experimental doses for the two modulators because these concentrations gave submaximal but substantial effects on the wild-type CaR. Using these two doses, we found that the efficacies of the two modulators were quite different depending on the mutant CaR that was tested. In most cases, NPS R-467 gave more pronounced changes in maximal response and EC 50 than did L-phenylalanine. In fact, there were several mutant CaRs that were responsive to NPS R-467 but not to L-phenylalanine. The most obvious exception to this observation is the wild-type CaR, which shows an unexplained decline in its maximal activity in the presence of NPS R-467. In addition, there were also several mutants that appeared to be more responsive to L-phenylalanine such as S147A and E297K. No mutant receptors were found to be responsive to L-phenylalanine but were found to be insensitive to NPS R-467. This difference in the efficacies of NPS R-467 and L-phenylalanine with mutant receptors suggests separate sites of action for these two allosteric modulators. Corroborating this point was the synergistic behavior of NPS R-467 and L-phenylalanine. Each modulator had additional potentiating effects on some mutant receptors in the presence of a maximal concentration of the other modulator.
NPS R-467 can be effective with mutant CaRs that are not normally responsive to Ca 2ϩ o . For example, the responsiveness of G143E is restored to 26% of that of the wild-type receptor in the presence of 1 M NPS R-467, whereas the mutant receptor is completely inactive in the absence of NPS R-467 (see Fig. 4). In contrast, L-phenylalanine appears to need some degree of Ca 2ϩ o responsiveness to positively modulate the activity of CaR as suggested by the lack of positive modulation of G143E and G549R by L-phenylalanine. The difference may be attributed to the mechanism of action of NPS R-467, which may allow it to magnify subthreshold activation of the CaR. L-Phenylalanine had little effect on Hill coefficients of the wild-type and mutant CaRs, whereas NPS R-467 often had a negative impact on the Hill coefficients of the wild-type and some mutant receptors (Table I). These findings suggest that the action of NPS R-467 appears to be less dependent on the Ca 2ϩ o binding sites. It is probable that the interaction of the CaR with NPS R-467 increases the affinity of the receptor for its cognate G proteins or enhances signal transduction from the head of the CaR to its intracellular domains. When taken together with previous studies on the probable binding sites of NPS R-467 and L-phenylalanine, our data are consistent with the findings that NPS R-467 acts at the transmembrane region of the CaR (3).
In conclusion, these two types of allosteric modulators act on the receptor not only through distinct sites but also through distinct mechanisms. Our findings provide a solid base for developing novel therapeutic agents that interact with the CaR with high affinities through the amino acid binding site. In addition, the synergistic effect of these two types of allosteric modulators on the CaR potentially provides a new therapeutic approach to the management of severe hypercalcemia associated with various types of hyperparathyroidism.