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J. Biol. Chem., Vol. 277, Issue 48, 46622-46631, November 29, 2002

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Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca²⁺ Receptor Critical for Response to Ca²⁺ and a Positive Allosteric Modulator^*

Jianxin Hu^§, Guadalupe Reyes-Cruz, Wangzhong Chen^¶, Kenneth A. Jacobson^¶, and Allen M. Spiegel

From the  Molecular Pathophysiology Section, NIDCD, and the ^¶ Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 16, 2002, and in revised form, September 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca²⁺ receptor (hCaR) in receptor activation by Ca²⁺ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca²⁺, and on NPS R-568 augmentation of sensitivity to Ca²⁺. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp⁷⁵⁸, Glu⁷⁵⁹, and Glu⁷⁶⁷, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca²⁺. Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca²⁺. Mutation of Glu⁸³⁷ in exo-loop 3 to alanine did not alter Ca²⁺ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu⁸³⁷ drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu⁸³⁷ at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The G protein-coupled [Ca²⁺]_o receptor (CaR)¹ plays a central role in the regulation of [Ca²⁺]_o homeostasis (1, 2). [Ca²⁺]_o activates the CaR in the parathyroid, thereby inhibiting parathyroid hormone secretion, and in the kidney, causing increased urinary calcium excretion. The physiological importance of the CaR in determining the level at which [Ca²⁺]_o is set in vivo has been documented by the identification of inactivating mutations in the CaR gene as the cause of familial hypocalciuric hypercalcemia and activating mutations as the cause of autosomal dominant hypocalcemia (3, 4). Naturally occurring CaR mutations identified in subjects with autosomal dominant hypocalcemia generally cause increased CaR sensitivity to [Ca²⁺]_o rather than causing constitutive activation (5).

The CaR belongs to a unique subfamily, family 3, of G protein-coupled receptors (GPCR) with an unusually large N-terminal, extracellular domain (ECD) comprised of Venus's-flytrap (VFT) and cysteine-rich domains, in addition to the seven-transmembrane domain (7TM) characteristic of all GPCR (6). Studies with chimeric family 3 GPCR (7-10) and the three-dimensional structure of the metabotropic glutamate type 1 receptor (mGluR1) determined by x-ray crystallography (11) show that the VFT is the site of agonist binding in family 3 GPCR. The precise site(s) for binding to the CaR, however, have not been identified.

The activation of family 3 GPCR can also be positively modulated by compounds that bind to the 7TM domain and are presumed to act allosterically (12). The phenylalkylamine, NPS R-568, a so-called calcimimetic, increases the sensitivity of the receptor to [Ca²⁺]_o activation (13), acting as a positive allosteric modulator by binding to the CaR 7TM region (14). A CaR mutant lacking the ECD that responds minimally to [Ca²⁺]_o shows significant responsiveness when NPS R-568 is added (14). This suggests that sites involved in [Ca²⁺]_o activation are present within the 7TM domain and not only the ECD. Stretches of acidic residues within the VFT have been considered likely sites of Ca²⁺ binding to the CaR (15). Given that the 7TM domain also is capable of being activated by [Ca²⁺]_o, we decided to investigate the role of acidic residues located in the extracellular loops of the 7TM domain in this activation. The exo-loops of the 7TM domain, like the ECD, are presumptively exposed at the cell surface and therefore could be involved in extracellular Ca²⁺ binding to and activation of the CaR.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Site-directed Mutagenesis of the hCaR-- The full-length hCaR cDNA cloned in the pCR3.1 expression vector and a truncated receptor lacking the ECD, Rho-C-hCaR, were described previously (5, 16). Rho-C-hCaR contains the first 20 amino acids of the N terminus of bovine rhodopsin fused to amino acid residues 600-903² of the wild type hCaR (see Fig. 1). Site-directed mutagenesis was performed using the QuikChange^TM site-directed mutagenesis kit (Stratagene Inc., La Jolla, CA), according to the manufacturer's instructions. Parental hCaR cDNA in pCR3.1 vector was amplified using Pfu Turbo DNA polymerase with mutagenic oligonucleotide primers (sequences available on request) for 16 cycles in a DNA thermal cycler (PerkinElmer Life Sciences). After digestion of the parental DNA with DpnI for 1 h, the amplified DNA with incorporated nucleotide substitution was transformed into Escherichia coli (DH-5 strain). The sequence of mutant receptors was confirmed by automated DNA sequencing using a dRhodamine Terminator Cycle sequencing kit and ABI PRISM-373A DNA sequencer (PE Applied Biosystems, Foster City, CA).

Transient Transfection of Wild Type and Mutant Receptors in HEK-293 Cells-- Transfections were performed using 12 µg of plasmid DNA for each transfection in a 75-cm² flask of HEK-293 cells. DNA was diluted in serum-free DMEM (BioFluids Inc., Rockville, MD), mixed with diluted LipofectAMINE (Invitrogen), and the mixture was incubated at room temperature for 30 min. The DNA-LipofectAMINE complex was further diluted in 6 ml of serum-free DMEM and was added to 80% confluent HEK-293 cells plated in 75-cm² flasks. After 5 h of incubation, 15 ml of complete DMEM containing 10% fetal bovine serum (BioFluids Inc.) was added. 24 h after transfection, the transfected cells were split and cultured in complete DMEM.

Phosphoinositide Hydrolysis Assay-- PI hydrolysis assay has been described previously (16). Briefly, 24 h after transfection, transfected cells from a confluent 75-cm² flask were split. Typically one-eighth of cells were plated in one well in a 6-well plate, and whole cell lysate was prepared 48 h post-transfection for Western blot assay. The remaining cells were plated in two 12-well plates in complete DMEM containing 3.0 µCi/ml of [³H]myo-inositol (PerkinElmer Life Sciences) and cultured for another 24 h. Culture medium was replaced by 1× PI buffer (120 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl₂, 20 mM LiCl in 25 mM PIPES buffer, pH 7.2) and incubated for 1 h at 37 °C. After removal of PI buffer, the cells were incubated for an additional 1 h with different concentrations of Ca²⁺ in 1× PI buffer with or without 1 µM calcimimetic compound NPS R-568 or NPS S-568. The reactions were terminated by the addition of 1 ml of acid-methanol (1:1,000 v/v) per well. Total inositol phosphates were purified by chromatography on Dowex 1-X8 columns, and the radioactivity for each sample was counted with liquid scintillation counter.

Graphs of concentration dependence for stimulation of PI hydrolysis by [Ca²⁺]_o for each transfection were drawn by using GraphPad Prism version 2.0 software. Each value on a curve is the mean of duplicate determinations unless otherwise indicated. The graphs shown in this paper are representative ones from at least three independent experiments.

Synthetic Chemistry-- The method for the synthesis of NPS R-568 was reported previously (14). For the synthesis of NPS S-568, racemic 1-(3-methoxyphenyl)ethylamine was prepared by reductive amination of 3-methoxyacetophenone with ammonium acetate (17), and the enantiomerically pure S-isomer was isolated through crystallization with S-mandelic acid (18). The final products were then obtained by reductive amination of 3-(2-chlorophenyl)propionaldehyde with the free base of S-1-(3-methoxyphenyl)ethylamine using sodium cyanoborohydride in tetrahydrofuran containing a trace of acetic acid to provide S-568. The amine was isolated as the hydrochloric salt following treatment of the free base with anhydrous hydrochloric acid in dioxane. The specific rotations, proton NMR spectra and high resolution mass spectra were consistent with the assigned structure.

Immunoblotting and Immunocytochemistry-- Confluent cells in 6-well plates were rinsed with ice-cold phosphate-buffered saline and scraped on ice in lysis buffer containing 20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 1% Triton X-100, and freshly added protease inhibitors mixture (Roche Molecular Biochemicals). For immunoblotting of full-length receptors, 50 µg of protein/lane reduced with -mercaptoethanol (5%) was separated on 5% SDS-PAGE gel. The proteins on the gel were electrotransferred onto nitrocellulose membrane and incubated with 0.1 µg/ml of protein A-purified mouse monoclonal anti-hCaR antibody ADD (raised against a synthetic peptide corresponding to residues 214-235 of hCaR protein). Subsequently, the membrane was incubated with a secondary goat anti-mouse antibody conjugated to horseradish peroxidase (Amersham Biosciences) at a dilution of 1: 2,000. The hCaR protein was detected with an ECL system (Amersham Biosciences). For immunoblotting of Rho-C-hCaR constructs, 20 µg of protein/lane premixed with 5% -mercaptoethanol for 1 h was separated on precast 4-20% gradient gels (Invitrogen), and mouse monoclonal anti-rhodopsin N terminus antibody B6-30, which was kindly provided by Paul Hargrave (University of Florida), was used as the primary antibody.

For fluorescence immunocytochemistry, transfected cells grown on duplicate coverslips precoated with 20 µg/µl Fibronectin (Calbiochem, La Jolla, CA) from human plasma were fixed with paraformaldehyde (4%) in Dulbecco's phosphate-buffered saline (BioFluids Inc.) for 20 min with one set of the coverslips permeabilized with methanol (100%). After 1 h of incubation at 37 C with mouse anti-rhodopsin N terminus antibody B6-30, the cells were washed and incubated with fluorescein-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Inc., West Grove, PA) for 45 min at room temperature. After washing, the cells on coverslips were mounted on glass slides using ProLong Antifade (Molecular Probes Inc., Eugene, OR) and examined by two independent observers, who assessed staining patterns without knowledge of the identity of cDNAs transfected, with a fluorescent microscope (Zeiss Axiophot, Zeiss, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcium Response by Full-length hCaR and ECD Deletion Mutant, Rho-C-hCaR-- We previously described construction of a mutant hCaR truncated at residue 903 and with a deletion of most of the ECD (residues 1-599). The first 20 amino acids of bovine rhodopsin were fused to hCaR residue 600 to facilitate cell surface expression of this mutant construct designated Rho-C-hCaR (Fig. 1) (5, 19). We transfected wild type and Rho-C-hCaR cDNAs into HEK-293 cells and analyzed their function by [Ca²⁺]_o-stimulated PI hydrolysis assay and their expression on immunoblots stained with either anti-hCaR monoclonal antibody ADD to detect full-length CaR or anti-rhodopsin N terminus antibody B6-30 to detect Rho-C-hCaR. Fig. 2 shows that wild type hCaR responds to [Ca²⁺]_o with an EC₅₀ value of 3.08 ± 0.04 mM (mean ± S.E., n = 5). Under reducing conditions, ADD antibody detected two major bands of about 130 and 150 kDa (Fig. 2). Previous studies have shown that the monomeric ~150-kDa band represents hCaR forms expressed at the cell surface and modified with N-linked, complex carbohydrates; the ~130-kDa band represents high mannose-modified forms, trapped intracellularly and sensitive to endoglycosidase H digestion (3, 16, 20)

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Schematic diagram showing amino acid sequence of the wild type hCaR and Rho-C-hCaR. The location of signal peptide, glycosylation sites, the sequence of synthetic polypeptide used to raise monoclonal antibody ADD, the beginning and end of the VFT domain, and residue Phe788 in TM5 are indicated. All eight acidic residues in the extracellular loops (exo-loops 1-3) are indicated by residue numbers and shown as white letters in black circles. The sequence of the first 20 amino acids of bovine rhodopsin N terminus fused to the hCaR beginning at residue 600 and ending at residue 903 comprising Rho-C-hCaR is also indicated.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration dependence for [Ca2+]o stimulation of PI hydrolysis in the presence or absence of 1 µM NPS R-568 (left panel) and immunoblot of CaR (right panel) in transiently transfected HEK-293 cells expressing WT hCaR, empty vector, Rho-C-hCaR, and Rho-C-hCaR/F788C. Transfection, PI assay, SDS-PAGE, and immunoblot with monoclonal anti-hCaR antibody ADD or monoclonal anti-rhodopsin N terminus antibody B6-30 were performed as described under "Materials and Methods." Molecular mass standards are indicated at the right of the blots. Left panel, results of PI assay are expressed as percentages of maximal response (wild type hCaR at 8 mM). Right panel, immunoblot (ADD blot for full-length receptor and Rho-C blot for Rho-C-hCaR) of whole cell lysate of transfected HEK-293 cells. The immunoblots shown here and in Figs. 3-6 and 8 were done using cells from the same transfection as the cells used for PI hydrolysis assay. WT, wild type.

Rho-C-hCaR showed minimal response to [Ca²⁺]_o in the PI hydrolysis assay, suggesting that the ECD of the hCaR plays a critical role in ligand binding and activation of the receptor. Rho-C-hCaR, however, is capable of responding to [Ca²⁺]_o upon addition of 1 µM NPS R-568. This calcimimetic compound did not directly activate the receptor but markedly potentiated Ca²⁺-stimulated responses not only of the wild type hCaR but also of Rho-C-hCaR (Fig. 2). The addition of 1 µM NPS S-568 has no effect on the response to [Ca²⁺]_o of either WT hCaR or Rho-C-hCaR (data not shown). We found that imposing an activating mutation, F788C, identified in a subject with autosomal dominant hypocalcemia (21) onto Rho-C-hCaR slightly increased the basal activity of the mutant receptor and mimicked the effect of NPS R-568 in potentiating the [Ca²⁺]_o response. Immunoblot of Rho-C-hCaR constructs shows a single band at ~36 kDa for the monomeric form of the mutant receptor (Fig. 2, right panel). The immunoblot confirms the expression of Rho-C-hCaR constructs but does not distinguish between receptor proteins expressed at the cell surface and those retained intracellularly. The ability of Rho-C-hCaR to respond to extracellular calcium, however, indicates that at least some, if not all, of the receptor protein is expressed at the cell surface.

Assay of Mutant hCaRs with Acidic Residues in the Extracellular Loops Substituted by Alanine-- Although there is strong evidence that the VFT domain of the ECD of family 3 GPCRs, including the CaR, is responsible for agonist binding (7-10), the ability of Rho-C-hCaR to respond to calcium upon addition of NPS R-568 or by imposing the F788C mutation indicates the existence of calcium-binding site(s) in the 7TM domain of the hCaR as well. Acidic residues in the VFT have been speculated to be involved in calcium binding to and activation of the CaR. This focused our attention on the eight acidic residues in the extracellular loops of the 7TM domain of the CaR (two in exo-loop 1, five in exo-loop 2, and one in exo-loop 3; Fig. 1) to assess their possible role in the activation of the receptor by calcium. We constructed by site-directed mutagenesis mutant full-length hCaRs with a single alanine mutation substituted for each of the respective acidic residues in exo-loops 1-3. Fig. 3 shows that the eight different mutants were expressed in the same pattern of two major bands as the wild type, indicating that the hCaR is tolerant of single alanine substitution in these sites without abolishing cell surface expression. The density of upper bands of these mutants varied slightly. Our recent study showed that different cell surface expression levels of hCaRs affect the maximal response of the receptor to [Ca²⁺]_o but not the EC₅₀ value of the receptor (22). Fig. 3 shows that three of the eight mutants, namely hCaR/D758A, hCaR/E759A, and hCaR/E767A, exhibited increased sensitivity to [Ca²⁺]_o. The EC₅₀ values for these activating mutants are 0.95 ± 0.05 mM (hCaR/D758A), 0.74 ± 0.02 mM (hCaR/E759A), and 1.83 ± 0.03 mM (hCaR/E767A) (mean ± S.E.; n = 3). The maximal activation by calcium for mutants D758A and E759A was 50-60% of that of the WT hCaR. In contrast, alanine substitution for each of the other five acidic residues did not change the sensitivity to calcium and in most cases minimally altered maximal response. E837A showed the lowest maximal response (~70% of WT) of these five mutants.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Concentration dependence for [Ca2+]o stimulation of PI hydrolysis (left panel) and ADD blot of CaR (right panel) in transiently transfected HEK-293 cells expressing WT hCaR or mutant CaRs with alanine substituted individually for each of the eight acidic residues in extracellular loops 1-3. The methods and format for presentation of results are as described in the legend to Fig. 2 except that the maximal response is wild type (WT) hCaR at 30 mM.

Because alanine substitution for several individual acidic residues within exo-loop 2 led to increased sensitivity of the CaR, we created additional mutants substituting alanine for multiple acidic residues in exo-loop 2 to determine whether this would lead to further increases in CaR sensitivity. As shown in Fig. 4, a receptor with the cluster of four adjacent acidic residues Glu⁷⁵⁵, Glu⁷⁵⁷, Asp⁷⁵⁸, and Glu⁷⁵⁹ mutated simultaneously to alanines (termed hCaR/4A) showed increased sensitivity to [Ca²⁺]_o but only to a similar extent as the individual E767A mutant. However, changing all five acidic residues in exo-loop 2 to alanines simultaneously (termed hCaR/5A) not only did not lead to further increase in CaR sensitivity but instead resulted in significantly impaired function of the receptor with maximal activation <20% of wild type hCaR (Fig. 4). As shown by the faint upper band on immunoblot of the 5A mutant (Fig. 4, right panel), the loss of function of the 5A mutant is due to poor cell surface expression.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Concentration dependence for [Ca2+]o stimulation of PI hydrolysis (left panel) and ADD blot of CaR (right panel) in transiently transfected HEK-293 cells expressing WT hCaR, empty vector, hCaR/E767A, hCaR/4A (Glu755, Glu757, Asp758, and Glu759, all mutated to Ala), and hCaR/5A (all five acidic residues in extracellular loop 2 mutated to Ala). The methods and format for presentation of results are as described in the legend to Fig. 2 except that the maximal response is wild type (WT) hCaR at 30 mM.

We extended our study of exo-loop 2 alanine mutants by combining them with the naturally occurring F788C mutation (hCaR/F788C) identified in subjects with autosomal dominant hypocalcemia (21). In vitro study shows that this mutation causes a left shift in the response of the receptor to [Ca²⁺]_o (Fig. 5). The F788C mutant receptor retains increased sensitivity to [Ca²⁺]_o when it is combined with the E767A mutation (hCaR/F788C/E767A) or 4A mutations (hCaR/F788C/4A), although maximal receptor activation is reduced. However, combining the hCaR/F788C mutant with the 5A mutations (hCaR/F788C/5A) resulted in complete loss of function in a [Ca²⁺]_o-stimulated PI hydrolysis assay (Fig. 5). Loss of receptor function was due to a lack of cell surface expression as reflected in absence of the upper band on immunoblot (Fig. 5, right panel).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Concentration dependence for [Ca2+]o stimulation of PI hydrolysis (left panel) and ADD blot of CaR (right panel) in transiently transfected HEK-293 cells expressing WT hCaR, hCaR/F788C, hCaR/F788C/E767A, hCaR/F788C/4A, and hCaR/F788C/5A. The methods and format for presentation of results are as described in the legend to Fig. 2.

Assay of Mutant Rho-C-hCaRs with Acidic Residues in the Extracellular Loops Substituted by Alanines-- We next tested the effects of alanine substitution of acidic residues in the exo-loops of Rho-C-hCaR that lacks the ECD. Mutant receptor function was tested by using the intact cell [Ca²⁺]_o-stimulated PI hydrolysis assay with and without the addition of 1 µM NPS R-568. Fig. 6A shows that without addition of NPS R-568, most receptor constructs showed minimal response to [Ca²⁺]_o, even up to 30 mM. The E767A mutation, and to a much lesser extent, the 4A mutation revealed a significant [Ca²⁺]_o response within the Rho-C-hCaR context. With the addition of 1 µM NPS R-568, as shown before, Rho-C-hCaR was significantly activated by [Ca²⁺]_o. With NPS R-568, the E767A and 4A mutants showed greater activation by [Ca²⁺]_o than Rho-C-hCaR, as did the E671A mutant (Fig. 6B). The D674A mutant was also activated by [Ca²⁺]_o in the presence of 1 µM NPS R-568 but to a lower extent than Rho-C-hCaR. Importantly, two mutant constructs, Rho-C-hCaR/5A and Rho-C-hCaR/E837A, exhibited no response to [Ca²⁺]_o even in the presence of 1 µM NPS R-568. Studies with full-length hCaR/5A mutant showed that it is very poorly expressed at the cell surface, thus the lack of response of Rho-C-hCaR/5A could also reflect poor cell surface expression. Full-length hCaR/E837A mutant, however, is expressed at the cell surface and is clearly activated by [Ca²⁺]_o. Thus the lack of [Ca²⁺]_o activation of the Rho-C-hCaR/E837A mutant, even with addition of NPS R-568, was unexpected. Immunoblotting showed that all Rho-C-hCaR constructs are expressed, but unlike with full-length CaR, immunoblotting alone does not distinguish between cell surface-expressed and intracellular forms of receptor. Therefore additional approaches were needed to assess cell surface expression of the Rho-C-hCaR mutants, particularly Rho-C-hCaR/5A and Rho- C-hCaR/E837A.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Concentration dependence for [Ca2+]o stimulation of PI hydrolysis in the absence (A) or presence of 1 µM NPS R-568 (B) and Rho-C blot of CaR (C) in transiently transfected HEK-293 cells expressing Rho-C-hCaR, empty vector, Rho-C-hCaR/E767A, Rho-C-hCaR/4A, Rho-C-hCaR/5A, Rho-C-hCaR/E671A, Rho-C-hCaR/D674A, and Rho-C-hCaR/E837A. The methods and format for presentation of results are as described in the legend to Fig. 2 (percentage of maximal response Rho-C-hCaR at 30 mM + NPS R-568).

Immunocytochemistry of Cells Expressing Rho-C-hCaR/5A and Rho-C-hCaR/E837A-- To document whether the Rho-C-hCaR/5A and Rho-C-hCaR/E837A mutants were expressed at the cell surface, immunocytochemistry was performed with the anti-rhodopsin N terminus antibody B6-30, the epitope of which is located in the extracellular N terminus. Fig. 7 shows that HEK-293 cells transfected with wild type Rho-C-hCaR or Rho-C-hCaR/E837A were stained by antibody B6-30 both under nonpermeabilized and permeabilized conditions, indicating successful cell surface expression of these receptors. In contrast, cells transfected with Rho-C-hCaR/5A were stained only under permeabilized conditions (Fig. 7), indicating that Rho-C-hCaR/5A was expressed intracellularly only.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Fluorescence immunocytochemistry, under nonpermeabilized (A) or permeabilized (B) conditions, of cells transfected with cDNAs encoding Rho-C-hCaR, Rho-C-hCaR/5A, Rho-C-hCaR/E837A, and vector only. Transfection, immunostaining with mono clonal anti-rhodopsin N terminus antibody B6-30, and fluorescence microscopy were performed as described under "Materials and Methods." Phase contrast images for Rho-C-hCaR/5A and vector transfected cells under nonpermeabilized conditions are shown to indicate the presence of cells.

Assay of Response to NPS R-568 by hCaR/E837A-- Given that the Rho-C-hCaR/E837A mutant is expressed at the cell surface and yet does not respond to [Ca²⁺]_o in the presence of NPS R-568, we considered the possibility that Glu⁸³⁷ may be involved in the action of NPS R-568 on the hCaR and that mutating this residue to alanine impairs NPS R-568 modulation of hCaR activation. To test this possibility, we measured [Ca²⁺]_o-stimulated PI hydrolysis of cells transfected with hCaR/E837A in the presence or absence of 1 µM NPS R-568, comparing it with two other alanine mutants, hCaR/E757A and hCaR/E767A. Fig. 8A shows that NPS R-568 failed to potentiate the response of hCaR/E837A to [Ca²⁺]_o, whereas the calcimimetic compound significantly potentiated the response of hCaR/E757A to [Ca²⁺]_o and even further potentiated the already left-shifted [Ca²⁺]_o response of hCaR/E767A. To evaluate further the effect of the E837A mutation on responsiveness to NPS R-568, we tested a range of concentrations of NPS R-568 up to 100 µM in the PI hydrolysis assay at 2 mM [Ca²⁺]_o. Fig. 8B shows that concentrations as low as 0.1 µM NPS R-568 enhanced the WT hCaR response to [Ca²⁺]_o and that the effect of NPS R-568 reached a maximum at 10 µM. In contrast, the hCaR/E837A mutant response to NPS R-568 was significantly right-shifted. No significant enhancement of [Ca²⁺]_o response by the hCaR/E837A mutant was observed unless the concentration of NPS R-568 was 10 µM or greater. Even at maximally effective concentrations of NPS R-568, the enhancement of [Ca²⁺]_o response of the hCaR/E837A mutant was less than 50% of that seen with WT hCaR. This difference in NPS R-568 effect was not a function of differences in receptor expression as seen on immunoblot (Fig. 8B, right panel).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   A, concentration dependence for [Ca2+]o stimulation of PI hydrolysis in the absence or presence of 1 µm NPS R-568 (left panel) and ADD blot of CaR (right panel) in transiently transfected HEK-293 cells expressing hCaR/E757A, hCaR/E767A, and hCaR/E837A. The methods and format for the presentation of results are as described in the legend to Fig. 2 except that the maximal response is hCaR/E757A at 30 mM without NPS R-568. B, Concentration dependence for NPS R-568 stimulation of PI hydrolysis at 2 mM [Ca2+]o (left panel) and ADD blot of CaR (right panel) in transiently transfected HEK-293 cells expressing wild type (WT) hCaR and hCaR/E837A. The results of PI assay are expressed as percentages of maximal response (wild type hCaR at 50 µM NPS R-568). The results are the means ± S.E. of triplicate determinations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We tested the hypothesis that acidic residues in the extracellular loops of the 7TM domain of the hCaR are involved in [Ca²⁺]_o activation of the receptor by replacing each of these residues with alanine and measuring the ability of the respective mutant receptors to respond to [Ca²⁺]_o. Alanine mutagenesis of extracellular loop acidic residues was done both in the context of the full-length receptor and in Rho-C-hCaR, a mutant receptor lacking most of the ECD. Of the eight acidic residues in exo-loops 1-3, we identified three, Asp⁷⁵⁸, Glu⁷⁵⁹, and Glu⁷⁶⁷, all in exo-loop 2, that based on the evidence summarized below appear to play important roles in [Ca²⁺]_o activation of the receptor. We identified another acidic residue, Glu⁸³⁷ in exo-loop 3, as critical for receptor responsiveness to the positive allosteric modulator, NPS R-568. Interestingly, only these four of the eight acidic residues in exo-loops 1-3 are identically conserved in all CaR species sequenced to date. Glu⁷⁵⁵ is conservatively substituted by aspartate, and Glu⁶⁷¹, Asp⁶⁷⁴, and Glu⁷⁵⁷ are nonconservatively substituted or deleted in some species (23-26).

Mutation of Asp⁷⁵⁸, Glu⁷⁵⁹, and Glu⁷⁶⁷ individually to alanine or in combination such as the 4A mutant (Glu⁷⁵⁵, Glu⁷⁵⁷, Asp⁷⁵⁸, and Glu⁷⁵⁹, all mutated to alanine) increased the sensitivity of the full-length hCaR to [Ca²⁺]_o. The 4A and E767A mutants in the context of Rho-C-hCaR, moreover, showed increased responses to [Ca²⁺]_o alone compared with Rho-C-hCaR and showed a further augmentation in response to NPS R-568 compared with the response elicited in Rho-C-hCaR by the combination of [Ca²⁺]_o and NPS R-568. By comparison, mutation of any of the remaining five acidic residues in exo-loops 1-3 had no major effect on the sensitivity of response of either full-length hCaR or Rho-C-hCaR to [Ca²⁺]_o.

Combined mutation of all five acidic residues in exo-loop 2, the 5A mutant, drastically impaired [Ca²⁺]_o response in the context of the full-length receptor and abolished [Ca²⁺]_o response in the context of the full-length receptor with the F788C mutation that enhances [Ca²⁺]_o sensitivity or in Rho-C-hCaR even when NPS R-568 was added. Immunoblot of the full-length hCaR/5A mutant and of the 5A mutant also containing the F788C substitution showed greatly diminished or absent expression, respectively, of the ~150-kDa band that we and others have shown corresponds to the fully processed, cell surface-expressed form of the CaR (3, 16, 20). Thus the lack of [Ca²⁺]_o response of the full-length 5A mutants reflects the failure of these mutants to be expressed at the cell surface and indicates that alanine substitution for all five exo-loop 2 acidic residues impairs normal folding and/or processing of the full-length CaR. Immunoblot with the anti-rhodopsin B6-30 antibody indicated that the Rho-C-hCaR/5A mutant protein is expressed in transfected HEK-293 cells, but because Rho-C-hCaR does not undergo the extensive glycosylation of the full-length hCaR, the immunoblot is inadequate to distinguish between cell surface-expressed versus intracellular forms. Immunocytochemistry performed with the same antibody under cell permeabilized and nonpermeabilized conditions showed clearly that the Rho-C-hCaR/5A mutant, unlike Rho-C-hCaR, is not expressed at the cell surface, again explaining the failure to respond to the combination of [Ca²⁺]_o and NPS R-568.

Ray and Northup (27) recently reported that HEK-293 cells transiently transfected with the identical mutant, Rho-C-hCaR/5A, show poor activation by metal ions and organic polycations in the presence of NPS-R568 but retain responsiveness to the combination of [Ca²⁺]_o and poly-L-arginine. They interpreted these results as evidence for a role of the five acidic residues in exo-loop 2 in CaR response to [Ca²⁺]_o and to NPS R-568, but because they failed to provide evidence for cell surface expression of the Rho-C-hCaR/5A mutant, this interpretation may be incorrect. Our results show that the 5A mutation both in the context of Rho-C-hCaR and of the full-length hCaR, which Ray and Northup did not study, leads to poor cell surface expression. We did not study the response of the mutants we made to organic polycations; hence we cannot explain the basis for preservation of the poly-L-arginine response of the Rho-C-hCaR/5A mutant observed by Ray and Northup, but this response is surprising given our demonstration that the Rho-C-hCaR/5A mutant is poorly expressed at the cell surface.

We observed that the Rho-C-hCaR/E837A mutant also failed to respond to [Ca²⁺]_o plus NPS R-568, but unlike the Rho-C-hCaR/5A mutant, immunocytochemistry clearly showed that the Rho-C-hCaR/E837A mutant is expressed at the cell surface. This prompted us to examine the response of the full-length hCaR/E837A mutant to [Ca²⁺]_o and NPS R-568. The response of this mutant to [Ca²⁺]_o was comparable in sensitivity and only moderately reduced in maximal activation by comparison with wild type hCaR. But in contrast to wild type hCaR and other exo-loop acidic residue mutants such as E757A and E767A, the E837A mutant failed to respond to 1 µM NPS R-568. The sensitivity of the mutant to and maximal activation by NPS R-568 were sharply reduced by comparison with wild type hCaR, despite comparable levels of expression. Without binding studies, we cannot conclude that Glu⁸³⁷ is directly involved in binding NPS R-568, but our data point to an important role for this acidic residue in exo-loop 3 in the action of this compound.

NPS R-568 has been shown to act as a positive allosteric modulator of the CaR (13), i.e. it does not itself activate the receptor but increases its sensitivity to agonists such as [Ca²⁺]_o. It acts selectively on the CaR and not on related family 3 GPCR such as the mGluRs, and its site of action has been localized to the 7TM domain of the receptor (8, 14). Positive allosteric modulators have been identified for other family 3 GPCR such as mGluR1 (12) and GABA_B receptor (28). The site of action for the latter was not defined, but for positive allosteric modulators of mGluR1, residues in the third and fifth transmembrane helices of the 7TM domain were shown to be critical for selective response (12). A compound acting as a noncompetitive antagonist of glutamate action on mGluR1 has also been identified (29). Two residues, Thr⁸¹⁵ and Ala⁸¹⁸ in human mGluR1b, were shown to be critical for selective response to the noncompetitive antagonist CPCCOEt. Interestingly, these residues are located at the junction between the extracellular surface and transmembrane helix seven, and depending on exact alignment, Thr⁸¹⁵ may be the residue in human mGluR1b equivalent to Glu⁸³⁷ in the hCaR. Positive allosteric modulators of family 3 GPCR have been speculated to act by facilitating formation of the active conformation of the ECD, whereas CPCCOEt has been speculated to act by blocking signal transmission from the ECD to the 7TM domain. The mechanism of action of these agents cannot be clarified, however, until their precise binding sites are identified and the fundamental mechanism for family 3 GPCR activation by agonists is defined.

Family 3 GPCR function as dimers (6), and x-ray crystallographic studies of the VFT domain of the mGluR1 show that agonist binding leads to closure of the VFT and to a conformational change in which one VFT domain rotates 70 degrees relative to the other VFT about an axis perpendicular to the dimer interface (11). How this rotation is transmitted through the cysteine-rich domain (to which the VFT is covalently linked) to the 7TM domain can be explained in at least two ways: (a) the direct contact model, in which one or more portions of the ECD directly bind to portion(s) of the 7TM domain, particularly the extracellular loops, and (b) the indirect or "peptide-linker" model, in which movement of the 7TM domains occurs because of torsion exerted through the movement of the linker between the VFT and first transmembrane domains (30). In either model, the presumption is that movement of transmembrane helices within and possibly between the two 7TM domains of the dimer is ultimately responsible for receptor activation.

The present data do not permit us to discriminate definitively between the direct contact and indirect models of receptor activation, but they focus attention on sites within the 7TM domain that may be critical in either case. Our data show that three acidic residues in exo-loop 2, Asp⁷⁵⁸, Glu⁷⁵⁹, and Glu⁷⁶⁷, all appear to be important for activation of the CaR. One possible role for these amino acids would be their involvement in directly binding Ca²⁺. Although considerable evidence points to the VFT as the primary site of Ca²⁺ binding (7-10), evidence for positive cooperativity in Ca²⁺ response is consistent with multiple sites for binding on the CaR, including the possibility of lower affinity sites within the 7TM domain (31). Our data showing that mutation of both Asp⁷⁵⁸ and Glu⁷⁵⁹ or of Glu⁷⁶⁷ to alanine facilitates activation of both full-length and ECD-deleted receptors rather than diminishing Ca²⁺ response argues against a role for these residues in directly binding Ca²⁺. Unfortunately, it was not possible to evaluate the effect on Ca²⁺ response of mutating all five acidic exo-loop 2 residues to alanine, because this change severely impaired cell surface expression of the receptor. An alternative explanation for the role of acidic residues in exo-loop 2 in CaR activation is that they are involved in maintaining an inactive conformation of the 7TM domain. Mutation of these residues to alanine may lead to a change in conformation of transmembrane helix 5 and/or 4 that facilitates receptor activation.

The Rho-C-hCaR, despite adequate cell surface expression, responds poorly to even high (>20 mM) [Ca²⁺]_o. With addition of NPS R-568, however, a dose-dependent activation by [Ca²⁺]_o is seen, albeit to levels only ~30% of that seen with the full-length CaR. The ability of this positive allosteric modulator to enhance not only the [Ca²⁺]_o response of the full-length receptor but also that of Rho-C-hCaR implies that its action cannot be limited to an alteration in the conformation of the ECD. One possibility consistent with our results is that binding of NPS R-568 within the 7TM domain alters the configuration of the transmembrane helices in a way that facilitates receptor activation. Naturally occurring activating mutations within the 7TM domain such as F788C in transmembrane helix five might act in the same way as NPS R-568.

Although the present results do not permit more than a speculative model of CaR activation, they do highlight the importance of acidic residues in exo-loop 2 in activation of the receptor by Ca²⁺ and of the cell surface-proximal portion of transmembrane helix seven in the action of the positive allosteric modulator NPS R-568. These results underscore the need for further structural information not only of the ECD but also of the 7TM domain of family 3 GPCR for their mechanism of activation to be understood.

	ACKNOWLEDGEMENTS

We are grateful to Paul Goldsmith for expert advice and to Kendra Jones for assistance with cell culture and transfections.

	FOOTNOTES

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

^§ To whom correspondence should be addressed: Bldg. 10, Rm. 8C-101, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-9212; Fax: 301-402-0374; E-mail: jianxinh@ intra.niddk.nih.gov.

Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.M207100200

² Rho-C-hCaR was incorrectly described by us previously (5) as fusing the N-terminal 20 residues of bovine rhodopsin to hCaR residue 599 rather than residue 600.

	ABBREVIATIONS

The abbreviations used are: CaR, extracellular Ca²⁺ receptor; hCaR, human extracellular Ca²⁺ receptor; GPCR, G protein-coupled receptor; mGluR1, metabotropic glutamate receptor type 1; PI, phosphoinositide; ECD, extracellular domain; 7TM, seven-transmembrane domain; VFT, Venus's-flytrap; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; exo-loop, extracellular loop; PIPES, 1,4-piperazinediethanesulfonic acid; NPS R-568, (R)-N-(3-methoxy--phenylethyl)-3-(2'-chlorophenyl)-1-propylamine hydrochloride; NPS S-568, (S)-N-(3-methoxy--phenylethyl)-3-(2'-chlorophenyl)-1-propylamine hydrochloride.

	REFERENCES

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