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J. Biol. Chem., Vol. 276, Issue 47, 44129-44136, November 23, 2001

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Amino Acids in the Cytoplasmic C Terminus of the Parathyroid Ca²⁺-sensing Receptor Mediate Efficient Cell-surface Expression and Phospholipase C Activation^*

Wenhan Chang, Stacy Pratt, Tsui-Hua Chen, Lilly Bourguignon, and Dolores Shoback

From the Endocrine Research Unit, Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121

Received for publication, May 26, 2001, and in revised form, August 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca²⁺] ([Ca²⁺]_o) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca²⁺]_o increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca²⁺]_o (up to 30 mM) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser⁸⁶⁶ and Val⁸⁹⁵ using tandem-Ala and single-site mutagenesis. Two point mutants (His⁸⁸⁰  Ala and Phe⁸⁸²  Ala CaR) showed 50-70% reductions in high [Ca²⁺]_o-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His⁸⁸⁰ and Phe⁸⁸² are situated in a putative -helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Changes in the extracellular [Ca²⁺] ([Ca²⁺]_o)¹ regulate parathyroid hormone (PTH) secretion and renal Ca²⁺ excretion by activating membrane Ca²⁺ receptors (CaRs). These receptors belong to family C of the G protein-coupled receptor superfamily. In response to high [Ca²⁺]_o or a variety of polyvalent cations, CaRs activate phospholipase C (PLC), inhibit cyclic AMP formation, and open nonselective cation channels in parathyroid cells and in heterologous expression systems (1-4). Like other G protein-coupled receptors in family C, metabotropic glutamate receptors (5, 6), the -aminobutyric acid receptor-B (7), and a large group of pheromone receptors (8, 9), the bovine parathyroid CaR has a large N-terminal extracellular domain (613 amino acids) and C-terminal tail (222 amino acids). Functional properties are beginning to be ascribed to specific domains of the CaR.

Considerable insights regarding the structure-function properties of the CaR have resulted from studying naturally occurring receptor mutants in kindreds with familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia (10). These studies strongly support the idea that the amino acids in the N-terminal extracellular domain of the CaR directly or indirectly participate in binding of Ca²⁺ or other cationic agonists to the receptor and in determining receptor sensitivity to ligand. (10). Studies in vitro on the biochemical properties of loss-of-function mutations in the CaR, in combination with receptor mutagenesis, indicate that specific residues in the second and third intracellular loops of the CaR are critical for PLC activation and efficient cell-surface expression (11).

Studies of C-terminally truncated CaRs indicate that most of the amino acids in the tail of the receptor are not directly involved in PLC activation and intracellular Ca²⁺ mobilization (12-14). This work, however, further suggests that a discrete domain in the tail (residues 874-888 and a specific Thr residue) is important in modulating receptor expression, signal transduction, and desensitization (12-14).

The importance of the C-terminal tail of the CaR to parathyroid function is highlighted by studies of patients with FHH and neonatal severe hyperparathyroidism (NSHPT) who have mutations in this region. In one kindred, an Alu-repetitive insertion was found in their CaR genes at residue 876 (13). This results in a receptor protein that terminates at amino acid 876 but to which is added a non-CaR peptide, Gln-Leu-Thr-Leu-Ser plus 29 Phe residues, encoded by the inserted Alu sequence. When CaR cDNA with the same Alu insertion is expressed in HEK-293 cells, the ability of high [Ca²⁺]_o to raise intracellular [Ca²⁺] is significantly impaired (to 10% or less of the wt CaR) (13). In an unusual kindred with features of both FHH and primary hyperparathyroidism, mutation of Phe⁸⁸¹ (Phe⁸⁸² in the bovine CaR) to Leu in the C-terminal tail results in defective signal transduction (15). In HEK-293 cells, this mutant receptor shows reduced intracellular Ca²⁺ responses to high [Ca²⁺]_o (15). Finally, a family reported with a large deletion in the tail of the CaR (amino acids 895-1075) has autosomal dominant hypocalcemia indicating a gain-of-function (16). When expressed in HEK-293 cells, this mutant CaR demonstrates enhanced cell-surface expression and increased sensitivity to high [Ca²⁺]_o (16). Overall, this work supports the importance of the tail of the CaR and accentuates its diverse roles.

The present studies were undertaken to identify residues in the tail of the CaR involved in regulating receptor expression and coupling to signaling pathways. Our results demonstrated that His⁸⁸⁰ and Phe⁸⁸², in a putative -helical domain in the C-terminal tail of the CaR, are important for the efficient targeting of the receptor to the cell surface. Mutations of these residues and or their deletion cause intracellular retention of the receptor and reduced PLC activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The bovine parathyroid CaR cDNA (1) was provided by Dr. Edward Brown (Harvard Medical School, Boston). The Chameleon double-stranded, site-directed mutagenesis kit and pBluescript II SK (pBS) were obtained from Stratagene (La Jolla, CA). pcDNA1/Amp and pCEP4 were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were from Stratagene (La Jolla, CA), Life Technologies, Inc., and Promega (Madison, WI). Rabbit anti-CaR antisera (21825A and 321113A) were raised against an extracellular and intracellular epitope of the bovine parathyroid CaR, respectively, and affinity-purified as described (3). Mouse anti-Grp78 (BiP) was purchased from StressGen Biotechnologies Corp (Victoria, British Columbia, Canada). Rat anti-CD44 was developed and characterized previously (17, 18). Fluorescein-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-rat or -mouse IgG for immunocytochemistry were obtained from Molecular Probes, Inc. (Eugene, OR). Endoglycosidase H (Endo-H) and peptide N-glycosidase F (PNG-F) were purchased from New England Biolabs (Beverly, MA) and Roche Molecular Biochemicals, respectively. Other supplies were from previously noted sources (3, 11, 19).

Mutagenesis and Subcloning of CaR cDNAs-- The truncated CaR cDNAs (CaR-(1-866)/pGEM-HE, CaR-(1-895)/pGEM-HE, and CaR-(1-929)/pGEM-HE) (Fig. 1a) were made by ligating PCR-generated fragments with premature stop codons inserted after the residues Ser⁸⁶⁶, Val⁸⁹⁵, and Gln⁹²⁹, respectively, into wt CaR/pGEM-HE DNA. The wt CaR/pGEM-HE was constructed as described previously (3) and used as the template in PCR reactions to generate truncated CaR cDNA fragments. The upper primer used in PCRs is derived from a sequence (5'-TGCAGATTGTCATCTGTGCC-3') at the 5'-end of a unique XhoI site (nucleotide 2762) in the bovine parathyroid CaR cDNA. The three lower primers were derived from the sequences flanking the corresponding truncation sites (T-866, 5'-TGGTCTAGAGTCAGGAAGGCTTGAAGAGGATGAT-3'; T895, 5'-TGGTCTAGAGTCAGACGTTGCTGCGGCGCAGCGT-3'; and T929, 5'-TGGTCTAGAGTCACTGCTGCTGAGGGAACGGGT-3'). Each of these primers introduces a stop codon and XbaI site at the 3'-end of the construct. After digestion with XhoI and XbaI, PCR fragments were ligated into wt CaR/pGEM-HE (3, 11, 20). Tandem-alanine (TA) and single-site mutant receptors in the background of wt CaR/pBS and CaR-(1-895)/pBS were made by PCR or site-directed mutagenesis using the Chameleon kit according to the manufacturer's instructions. Mutations were confirmed by automated DNA sequencing (Biomolecular Resource Center, University of California, San Francisco). Wt CaR/pBS was generated by ligating an SmaI fragment (nucleotides 248-3819) of the bovine parathyroid CaR cDNA into pBS (11). CaR-(1-895)/pBS was constructed by ligating an XhoI/XbaI fragment from CaR-(1-895)/pGEM-HE into precut wt CaR/pBS containing the rest of the receptor (11, 20).

Subcloning of mutant CaR constructs into pcDNA1/Amp for transient transfections was done by ligating the XhoI-XbaI fragments from CaR-(1-866)/pGEM-HE, CaR-(1-895)/pGEM-HE, CaR-(1-929)/pGEM-HE, wt CaR/pBS, or CaR-(1-895)/pBS containing the truncated, mutated, or wt sequences of the C-terminal tail of the receptor into an XhoI-XbaI fragment of wt CaR/pcDNA1/Amp with the remaining CaR sequence. Wt CaR/pcDNA1/Amp was made by ligating the 3619-base pair fragment of wt CaR/pBS into pcDNA1/Amp cut with NotI and HindIII restriction enzymes. To generate mutant CaR constructs for stable transfections, a KpnI-NheI fragment of the pGEM-HE construct containing the mutant CaR cDNA of interest was subcloned into pCEP4 as described previously (3).

InsP Assay-- Total InsP accumulation was assessed by labeling transfected HEK-293 cells with [³H]myoinositol (2 µCi/ml) for 18-24 h as detailed previously (11). We measured [³H]InsP accumulation in triplicate in the presence of LiCl (10 mM) after incubating cells for 60 min with different [Ca²⁺]_o at 37 °C. Total [³H]InsPs were analyzed by anion-exchange chromatography (3, 21). Results expressed as the average fold increase in total [³H]InsPs were calculated by dividing total [³H]InsPs produced by elevating [Ca²⁺]_o by basal [³H]InsPs at 0.5 mM Ca²⁺. All experiments were repeated at least 3 times for confirmation unless otherwise noted.

Immunoblotting and Immunocytochemistry-- Crude membrane protein fractions were prepared from HEK-293 cells, electrophoresed on SDS-PAGE gels, and transferred to nitrocellulose membranes as detailed previously (3, 19). Membranes were blotted with one of two affinity-purified rabbit antisera (21825B (50 nM) or 321113A (10 nM)) (3, 11, 22). Signal detection was by the enhanced chemiluminescence (ECL^TM) assay kit (Amersham Pharmacia Biotech). CaR protein expression in membrane protein fractions prepared from cells expressing wt and expressing mutant CaRs constructs was assessed by immunoblotting in at least two independent experiments.

In studies of receptor glycosylation, crude membrane protein fractions were digested with PNG-F or Endo-H as described (23). For experiments with Endo-H, membrane proteins (10 µg) were denatured in a solution containing SDS (0.05%, w/v) and 2-mercaptoethanol (50 mM) at 37 °C for 15 min. After adding Endo-H (500 units), NaOAc (70 mM; pH 5.2), and Triton X-100 (0.8%, v/v), proteins were further incubated at 37 °C for 1 h. For PNG-F digestion, membrane proteins (10 µg) were incubated with this enzyme (0.2 units) in the presence of Tris-HCl (150 mM; pH 8) and Triton X-100 (1.3%) at 37 °C for 1 h after samples were denatured with SDS (0.375%, w/v) and 2-mercaptoethanol (75 mM) at 37 °C for 15 min. Digested samples were immunoblotted as above.

For dual-fluorescence immunocytochemistry, transfected cells grown on coverslips were fixed with paraformaldehyde (4%) for 20 min and permeabilized with methanol (80%) (11). After overnight incubation at 4 °C with rabbit anti-CaR antiserum (21825A; 500 nM) plus either mouse anti-Grp78 (BiP) or rat anti-CD44 antiserum, cells were washed and incubated with fluorescein-conjugated and Texas Red-conjugated anti-IgG antibodies for 60 min at room temperature (3, 19). After washing, cells on coverslips were mounted on glass slides using Gel Mount (Biomeda, Foster City, CA) and examined with a Leica TCS confocal microscope (Laboratory for Cell Imaging, San Francisco Department of Veterans Affairs Medical Center). Fluorescent images were obtained sequentially and stored in a microcomputer. Paired pseudo-colored images and their overlays are presented.

Statistics-- Differences between wt and mutant CaR responses were tested by analysis of variance with the F-test using Excel 98 (MicroSoft, Seattle, WA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Signaling Properties and Expression of CaRs with Truncated C-terminal Tails-- To determine whether the C-terminal tail of the bovine parathyroid CaR plays a role in signal transduction, we constructed three truncated receptors, CaR-(1-866), CaR-(1-895), and CaR-(1-929), that retain 3, 32, or 66 of the 222 amino acids in the tail, respectively (Fig. 1a). Truncated and wt CaRs were either stably or transiently expressed in HEK-293 cells, and PLC activation was assessed by total InsP accumulation in response to changes in [Ca²⁺]_o from 0.5 to 5.0 mM. In cells stably expressing wt CaRs, this increment in [Ca²⁺]_o increased InsP production by 24.5 ± 2.8-fold (Fig. 1b), not significantly different from the responses in cells expressing CaR-(1-929) (28.8 ± 3.6-fold). In cells expressing CaR-(1-895), however, raising [Ca²⁺]_o from 0.5 to 5 mM increased InsPs by 10.7 ± 1.7-fold. This response was 40% that of wt CaR-expressing cells and was confirmed in three independent cell lines tested (Fig. 1b). In cells stably expressing CaR-(1-866) with only a three-amino acid tail, raising [Ca²⁺]_o from 0.5 to 30 mM failed to stimulate InsP production (Fig. 1b and data not shown). These findings indicate that progressive truncation of the C-terminal tail of the CaR reduced the ability of the receptor to activate PLC.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Signal transduction and expression of C-terminally truncated bovine parathyroid CaRs. a, schematic diagram of three truncated receptors: CaR-(1-866) (T866), CaR-(1-895) (T895), and CaR-(1-929) (T929). In this diagram, only the intracellular domains are represented. b, [3H]InsPs responses in HEK-293 cells stably expressing truncated mutants, wt CaR (WT), and vector DNA (V), after exposure to an increment in [Ca2+]o from 0.5 to 5.0 mM. [3H]InsPs were extracted and quantified as described under "Experimental Procedures." Results represent the average fold increase in total [3H]InsPs compared with basal levels at 0.5 mM Ca2+. c, membrane protein fractions (80 or 800 µg) from HEK-293 cells stably expressing the constructs noted were immunoblotted with anti-CaR antiserum (21825A, 50 nM) as described under "Experimental Procedures." d, [3H]InsPs production in HEK-293 cells transiently expressing truncated mutants, wt CaR (WT), and vector DNA (V). e, immunoblots of membrane protein fractions from HEK-293 cells transiently expressing the same cDNA constructs. f, confocal immunomicroscopy of transiently transfected cells expressing CaR truncation mutants and wt CaR (wt). Cells were stained with anti-CaR antiserum (21825A, 50 nM) and detected with fluorescence-conjugated goat anti-rabbit IgG antisera (left panels) and with anti-CD44 detected by goat anti-rat IgG antisera conjugated with Texas Red (middle panels). Overlay of double-stained cells are shown (right panels).

To determine whether the dampened signaling of these stably expressed truncated receptors was due to reduced expression, we performed immunoblotting. Approximately 10-fold more membrane protein from cells expressing CaR-(1-895) was required to produce a signal comparable with that of cells stably expressing wt CaRs (Fig. 1c). The level of expression of the CaR-(1-866) in HEK-293 cells was significantly higher than that of CaR-(1-895) (Fig. 1c). This was confirmed in two additional cell lines we generated and suggested that the inability of this mutant to activate PLC was not due to deficient receptor synthesis.

To address whether the above properties of truncated CaRs were in some way due to stable transfections, we compared these results to those from transiently transfected cells. High [Ca²⁺]_o-induced InsP responses in HEK-293 cells transiently expressing CaR-(1-895) were comparable with those of the wt CaR (Fig. 1d). As predicted, the expression levels of this mutant receptor were also comparable with those of the wt CaR (Fig. 1e). High [Ca²⁺]_o, however, also failed to increase InsP production in cells transiently expressing CaR-(1-866). The level of expression of CaR-(1-866) in these experiments was comparable with the wt CaR (Fig. 1e). In contrast to CaR-(1-895), the level of expression of the severely truncated mutant CaR-(1-866) was similar to that of wt CaRs in both transient and stable transfections.

Immunoblots showed the expected differences in size between wt and truncated CaRs as follows: wt CaR > CaR-(1-929) > CaR-(1-895) > CaR-(1-866) (Fig. 1, c and e). In both stably and transiently transfected cells expressing these cDNAs, two predominant glycosylated receptor forms as well as larger, likely aggregated receptors (>205 kDa) were evident (Fig. 1e). The expression pattern of CaR-(1-866) in stably transfected cells differed somewhat from that of transiently transfected cells. Although the 100-kDa protein was observed in both systems (Fig. 1, c and e), a 125-kDa band was seen only in transiently transfected cells (Fig. 1e), suggesting that post-translational modifications of this mutant receptor might differ as a result of variations in expression levels or receptor processing. This was confirmed in two immunoblots from two different membrane preparations.

To determine whether these truncated receptors were efficiently targeted to the membrane, we used confocal immunomicroscopy. In transiently transfected HEK-293 cells, CaR-(1-866), CaR-(1-895), and CaR-(1-929) were strongly localized to the membrane (Fig. 1f, green) and co-localized extensively with the hyaluronic acid receptor CD44, which serves as a cell-surface marker (17-18) (Fig. 1f, red). This was evident with the overlay of images (Fig. 1f, yellow). Thus, the signaling defects of CaR-(1-866) were clearly not the result of the inability of this receptor to be inserted into the membrane.

Signaling Properties and Expression of CaRs with TA or Single-site Mutations in the C-terminal Tail-- The above observations indicate that the region between residues 866 and 895 may contain critical determinants of PLC activation. To identify further the specific residues involved, we scanned this region by constructing seven TA mutant receptors in which 2-4 non-Ala residues were substituted with Ala, initially in the context of CaR-(1-895) (Fig. 2a). When these receptors were transiently expressed in HEK-293 cells, their ability to increase InsP production varied (Fig. 2b). The two most striking mutants (Ala^879-883 CaR-(1-895) and Ala^892-895 CaR-(1-895)) had at least a 50% reduction in their responses to a 4.5 mM increment in [Ca²⁺]_o (asterisks, Fig. 2, a and b). Their InsP responses were only 3.1 ± 0.4- and 2.8 ± 0.1-fold, respectively. This was 40-45% of the response in cells expressing CaR-(1-895) (7.1 ± 0.4-fold increase) (Fig. 2b). These findings indicate that mutating residues in these two regions significantly blocked the signaling ability of CaR-(1-895).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Signal transduction and expression of mutants of the C-tail of the CaR. a, schematic diagram of seven TA constructs (lanes 2-8) scanning amino acids 867-895 of the bovine parathyroid CaR. TA mutants were made in the context of CaR-(1-895) (T895-CaR) (lane 1). b, [3H]InsP production in HEK-293 cells transiently expressing vector DNA (V), wt CaR (Wt), CaR-(1-895) (T895-CaR) (lane 1), and seven TA mutants (lanes 2-8) in response to raising [Ca2+]o from 0.5 to 5.0 mM. Asterisks depict mutants with significantly reduced InsP production (i.e. <50% of wt CaR responses). c, membrane protein fractions (25 µg) from HEK-293 cells transiently expressing wt CaR (Wt), CaR-(1-895) (T895-CaR) (lane 1), and TA CaR constructs (lanes 2-8) were immunoblotted with anti-CaR antiserum (21825A, 50 nM).

We next examined the expression of these TA CaR mutants by immunoblotting (Fig. 2c). Whereas the lower 110-kDa bands in CaR-(1-895) and the seven TA mutants were reduced in intensity, compared with the lower band (140 kDa) in the wt CaR, the intensity of this band was in fact greatest in membrane fractions from cells expressing Ala^879-883 CaR-(1-895) and Ala^892-895 CaR-(1-895) (Fig. 2c, lanes 5 and 8). The intensity of the upper band (140 kDa) in membrane fractions from Ala^879-883 CaR-(1-895) and Ala^892-895 CaR-(1-895) mutant expressing cells was greater than in CaR-(1-895) (Fig. 2c, lanes 5 and 8 versus 1). Thus, it is unlikely that the signaling defects we observed in these two TA mutants are due to defective receptor synthesis.

To determine which residue(s) in these regions are responsible for reduced signaling through PLC, we engineered these TA mutants in the context of the full-length CaR. Surprisingly, in cells expressing Ala^892-895 CaR, raising [Ca²⁺]_o from 0.5 to 5.0 mM increased InsP production by 12.6 ± 1.3-fold (Fig. 3a). This was twice the response in cells expressing wt CaRs (6.0 ± 0.4-fold) in four experiments. The expression of Ala^892-895 CaR was comparable with that of wt CaR (Fig. 3b). Therefore, these sites were not examined further. In contrast, in cells expressing Ala^879-883 CaR, the ability of high [Ca²⁺]_o to increase InsPs was completely blocked (Fig. 3a), suggesting that residues 879-883 were required for efficient PLC activation. Because the expression level of Ala^879-883 CaR was comparable with that of the wt CaR (Fig. 3b), deficient receptor synthesis was judged not to be a likely explanation for the reduced signaling responses. Instead, these results suggested that one or more of three non-Ala residues, His⁸⁸⁰, Phe⁸⁸², and/or Lys⁸⁸³, are critical for efficient coupling of the CaR to PLC.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Signal transduction and protein expression of full-length CaRs with TA mutations. a, TA mutant CaRs (Ala879-883 CaR and Ala892-895 CaR) were generated as described under "Experimental Procedures." Mutant and wt CaR DNAs were transiently expressed in HEK-293 cells. Total [3H]InsPs were quantified and presented as Fig. 1. b, immunoblots of membrane proteins (25 µg) from HEK-293 cells transiently transfected with wt and TA mutant CaR cDNAs. Membrane protein fractions from vector cDNA-expressing cells showed no immunoreactivity (data not shown).

To pinpoint which of these three amino acids is important in mediating PLC signaling, we generated single-site Ala mutants (His⁸⁸⁰  Ala CaR, Phe⁸⁸²  Ala CaR, and Lys⁸⁸³  Ala CaR) and tested their ability to increase InsP production in response to increasing [Ca²⁺]_o from 0.5 to 5.0 mM. In cells expressing Lys⁸⁸³  Ala CaRs, this increment in [Ca²⁺]_o stimulated InsP production by 9.9 ± 0.3-fold, which was comparable with wt CaR responses (10.3 ± 0.9-fold). In cells expressing His⁸⁸⁰ Ala CaRs and Phe⁸⁸²  Ala CaRs, the same change in [Ca²⁺]_o increased InsP by 1.7 ± 0.1- and 3.6 ± 0.2-fold, which was only 17 and 35% of wt CaR responses, respectively (Fig. 4a). Dose-response studies showed that His⁸⁸⁰  Ala and Phe⁸⁸²  Ala CaR mutants had ED₅₀ values for Ca²⁺ that were substantially right-shifted to 8.5 ± 0.2 and 8.1 ± 0.2 mM Ca²⁺, respectively (p < 0.01, compared with an ED₅₀ value of 5.2 ± 0.2 mM Ca²⁺ for the wt CaR; see Fig. 4b). Maximal responses to 30 mM Ca²⁺ by His⁸⁸⁰  Ala and Phe⁸⁸²  Ala CaR mutants were also reduced to 5.7 ± 0.8- and 11.2 ± 0.8-fold, only 28 and 55% of the responses generated in cells expressing wt CaRs (20.5 ± 1.2-fold) (Fig. 4b). Levels of receptor expression for the wt and these mutant CaRs in crude membrane fractions were similar by immunoblotting and possibly even greater in cells expressing Phe⁸⁸²  Ala CaRs and Lys⁸⁸³  Ala CaRs (Fig. 4c). This suggested that the signaling defects of the His⁸⁸⁰  Ala and Phe⁸⁸²  Ala CaR mutants were not due to inadequate receptor synthesis.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Signal transduction and expression of CaRs with point mutations in the C-terminal tail. a, point mutations to Ala of three amino acids in the C-terminal tail were generated in the context of the full-length CaR as described under "Experimental Procedures." HEK-293 cells transiently transfected with these mutant constructs or the Ala879-883 CaR, wt CaR (WT), or vector cDNA were exposed to an increase in [Ca2+]o from 0.5 to 5.0 mM, and InsPs were determined. b, dose responses for [3H]InsP production with changing [Ca2+]o from 0.5 to 30 mM in cells transiently expressing vector (), wt CaR cDNA (), or mutant constructs (His880  Ala CaR () and Phe882  Ala CaR ()). c, immunoblots of membrane protein fractions (25 µg) from HEK-293 cells transiently expressing wt or mutant CaRs using anti-CaR antiserum (321113A, 10 nM).

Glycosylation of CaR Mutants-- CaRs are known to undergo post-translational modifications such as glycosylation that are required for efficient processing and eventually cell-surface expression (24). To test the possibility that alterations in glycosylation are responsible for the blunted PLC responses in CaR mutant-expressing cells, we examined the glycosylation patterns of wt and mutant CaRs. Membrane proteins from cells expressing these receptors were digested with PNG-F and Endo-H. Endo-H, which removes mannose from glycoproteins, reduced the size of the 140-kDa form of the CaR to 120 kDa but had no effect on the 160-kDa band (Fig. 5). An 80-kDa band, which reacted with anti-CaR antiserum, was also observed in the membrane proteins treated with Endo-H. Because the size of this band was significantly smaller than the predicted size of the CaR core protein, we speculate that it represents a degraded form of the CaR due to enzyme treatment. Digestion with PNG-F, which removes all N-linked carbohydrates, reduced the sizes of both the 140- and 160-kDa bands to 120 kDa (Fig. 5) as previously observed (12, 23). In cells transfected with Ala^879-883 CaR, His⁸⁸⁰  Ala CaR, and Phe⁸⁸²  Ala CaR cDNAs, both the 140- and 160-kDa forms of these receptors were present at levels comparable with those in cells expressing wt CaRs (Fig. 5). Both glycosylated forms of these mutant receptors were also sensitive to PNG-F digestion, and only the 140-kDa receptor was digested by Endo-H (Fig. 5). These results suggest that mutations at these sites do not change the glycosylation of CaRs, reflected by glycosidase sensitivity, and confirm the findings of Ray et al. (12) for a different panel of TA mutants encompassing sites within the tail of the human pituitary CaR.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   Deglycosylation of CaRs. Membrane proteins (10 µg) from HEK-293 cells expressing wt or mutant CaRs were digested with (+) or without () Endo-H or PNG-F and immunoblotted with anti-CaR antiserum (321113A, 10 nM) as described under "Experimental Procedures." Immunoblots are representative of two independent experiments.

Cell-surface Expression of Mutant CaRs-- To test the possibility that reduced signaling by Ala^879-883 CaR, His⁸⁸⁰  Ala CaR, and Phe⁸⁸²  Ala CaR is due to decreased cell-surface expression, we co-localized the CaR with the membrane receptor for hyaluronic acid CD44 (17). In cells transiently expressing wt CaR cDNA, receptors were detected in the membrane (Fig. 6, green). These receptors co-localized with CD44 (Fig. 6, red) as demonstrated by the overlay of signals yielding a yellow color. Levels of CD44 surface staining were equivalent in cells transfected with wt CaR, Ala^879-883 CaR, His⁸⁸⁰  Ala CaR, and Phe⁸⁸²  Ala CaR cDNAs (Fig. 6, red). A large portion of the mutant CaR proteins, in contrast to wt CaRs, was, however, localized to intracellular compartments that lacked CD44 staining (Fig. 6). Only a small proportion of total cellular CaR staining in cells expressing these mutants was present in the membrane. The minimal degree of overlapping signals argues against significant co-localization of CaR protein and CD44 in cells expressing these three mutants. These findings support the idea that the reduced ability of these mutant receptors to activate PLC is due to diminished cell-surface expression.

View larger version (98K): [in this window] [in a new window]   Fig. 6.   Dual-fluorescence immunocytochemistry of cells transiently expressing wt and mutant CaR cDNAs using anti-CaR (21825A) and anti-CD44 antisera and confocal microscopy. Expression of CaR and CD44 is indicated by the signals from fluorescein-conjugated (green) and Texas Red-conjugated (red) anti-IgG, respectively. Overlays of paired images for CaR and CD44 from the same cells are shown.

To localize further the intracellular compartments in which these mutant receptors reside, we performed immunocytochemistry using the ER marker BiP or Grp78 (25). In HEK-293 cells transfected with wt CaR cDNA, BiP staining was distributed predominantly in a punctate and in some areas in a mesh-like pattern. There was very little overlap with cell-surface staining of CaRs or staining in submembrane vesicles (Fig. 7, top panels). On the other hand, in cells expressing Ala^879-883 CaR, His⁸⁸⁰  Ala CaRs, or Phe⁸⁸²  Ala CaRs, the majority of CaR staining was intracellular. Much of it co-localized with BiP (Fig. 7). This confirmed the intracellular retention of these mutant CaRs. Thus, it appears likely that mutating His⁸⁸⁰ and Phe⁸⁸² either interferes with protein folding and or blocks CaR trafficking to the cell surface.

View larger version (75K): [in this window] [in a new window]   Fig. 7.   Dual-fluorescence immunocytochemistry of cells transiently expressing wt and mutant CaR cDNAs using anti-CaR (21825A) and anti-BiP antisera. Expression of CaR and BiP is indicated by the signals from fluorescein-conjugated (green) and Texas Red-conjugated (red) anti-IgG, respectively. Overlays of paired images for CaR and BiP from the same cells are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These studies suggest that a large portion of the C-terminal tail of the CaR is not required for PLC activation. The truncated receptors CaR-(1-929) and CaR-(1-895), expressed at levels comparable with the wt CaR, retain the ability to signal through PLC comparable with wt CaRs. This is consistent with a previous report (12) that deleting amino acids beyond residues 887 in the human CaR (compared with 888 in bovine parathyroid CaR) does not affect the ability of the receptor to activate PLC. We further found that deleting amino acids 867-895 in the C-terminal tail of the CaR completely blocked the ability of this truncated receptor to mediate high [Ca²⁺]_o-induced PLC responses despite strong membrane CaR expression. This suggested that important signaling determinants are present in this region of the CaR. Indeed, TA and single-site mutagenesis identified two amino acids, His⁸⁸⁰ and Phe⁸⁸², that turned out to be critical for efficient cell-surface expression of the full-length CaR and ultimately its ability to activate PLC maximally with the appropriate sensitivity to Ca²⁺. Mutating these residues to Ala caused significant intracellular retention of CaRs in transfected cells and reduced the ability of high [Ca²⁺]_o to stimulate PLC activity. Co-localization of these mutant receptors with the ER marker BiP suggested that their intracellular retention is likely due to defective receptor trafficking to the cell surface or enhanced degradation within cells.

BiP, a 78-kDa protein, is transiently associated with newly synthesized secretory and membrane proteins in the ER (25). It binds to improperly folded, incompletely glycosylated, or mutated proteins and prevents their trafficking (26-30). It is likely that mutating His⁸⁸⁰ and Phe⁸⁸² caused the intracellular retention of the resulting receptor proteins either by interrupting their folding or glycosylation. The glycosylation patterns of these mutant CaRs and their sensitivity to digestion by Endo-H and PNG-F, however, were indistinguishable from wt CaRs, suggesting that altered glycosylation does not explain the intracellular trapping of these mutants. These mutations, therefore, likely altered the conformation or folding of the resulting receptors.

Mutating amino acids in the C-terminal tail of the CaR may also block the interactions of receptor proteins with downstream signaling molecules. In cells expressing Ala^879-883 CaRs, high [Ca²⁺]_o was unable to increase InsP production, despite the presence of receptors on the cell surface. Ray et al. (12) showed that mutating residues 875-879 to Ala in the human CaR (equivalent to residues from 876-880 in the bovine parathyroid CaR) also blocked high [Ca²⁺]_o-induced PLC activation. This was seen even after levels of surface expression of the mutant receptors were adjusted to that of the wt CaR by increasing the quantity of cDNA transfected (12). Thus, these mutants are intrinsically unable to interact with signaling molecules, even when their cell-surface expression is deliberately increased.

Interestingly, in cells expressing the single-site mutants His⁸⁸⁰  Ala CaR and Phe⁸⁸²  Ala CaR, high [Ca²⁺]_o increased InsP production to levels 30-50% of the responses of wt CaRs, despite profound intracellular retention of these receptors and decreased cell-surface receptor expression. Whereas mutating these two sites individually appeared to alter the folding or secondary structure sufficiently to cause retention of these receptors, these mutations were not sufficient to block the receptor's interactions with signaling molecules that likely require other residues. When multiple sites were mutated in tandem, as in Ala^879-883 CaR, coupling to downstream effectors was more dramatically impaired.

To examine whether a specific secondary structure is present between residues 866 and 895 and required for the function of the CaR, we did computer modeling using the Chou-Fasman method (31). As illustrated in Fig. 8, amino acids 877-891 in the CaR are predicted to adopt an -helical structure that includes His⁸⁸⁰ and Phe⁸⁸². Substitutions of these residues with Ala do not affect the potential formation of this helical structure, according to the predictions by computer modeling (data not shown). Our findings suggest, however, that the specific functional groups in these His and Phe residues are required for the receptor to function properly, even in the presence of a putative -helix. Our observations with the truncated receptor (CaR-(1-866)) and studies of similarly truncated receptors by others (12, 13) indicate that deleting the region containing this putative -helix produces receptors that are unable to activate PLC but that are not retained intracellularly. Studies with human CaRs, truncated at residue 865 (866 in the bovine parathyroid CaR), indicate that these receptors are normally glycosylated (12). This further supports the idea that the inability of CaR-(1-866) to activate PLC is not likely to be due to improper glycosylation. Our scanning of the residues between 866 and 895 disclosed no amino acids (other than His⁸⁸⁰ and Phe⁸⁸²) that significantly altered the signaling of the CaR. Thus, we speculate that defective signal transduction by CaR-(1-866) may be due to the absence of the -helical structure including His⁸⁸⁰ and Phe⁸⁸².

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Predicted helical conformation of the juxtamembranous portion of the C-terminal tail of the CaR. Secondary structure was predicted according to Chou-Fasman method using PeptideStructure and plotted using PlotStructure modules of Wisconsin Package version 10 software (Genetics Computer Group, Madison, Inc., WI). The truncation site at residue 895 is indicated.

Unexpectedly, our studies indicated that mutations have different effects on truncated receptors compared with wt CaRs. In the context of CaR-(1-895), both mutants (Ala^879-883 CaR-(1-895) and Ala^892-895 CaR-(1-895)) demonstrated at least 50% reductions in their PLC responses to high [Ca²⁺]_o compared with wt CaRs. In the context of the full-length CaR, however, only the Ala^879-883 CaR showed reduced signaling. Surprisingly, the reduced PLC activation due to mutating residues 879-883 to Ala was greater in the context of the wt CaR (>95% inhibition) than in the context of CaR-(1-895) (55% inhibition). This suggests that there may be inhibitory or modulatory domains in the full-length CaR beyond residue 895 that interact with residues closer to the membrane. In addition, why mutating residues 892-895 in the context of the truncated CaR-(1-895) blocked high [Ca²⁺]_o-induced signal transduction but had no effect when the same mutation was made in the context of wt CaR remains unclear. It is likely that multiple as yet unknown and important regulatory domains exist in residues beyond 895 in the CaR tail that are involved in modulating receptor function.

Naturally occurring mutations in the C-terminal tail of the CaR in FHH, NSHPT, and autosomal dominant hypocalcemia (13, 15) confirm the importance of this receptor domain in physiologic functions. Patients with FHH or NSHPT have increased serum Ca²⁺ and PTH levels, due to a reduced ability of high [Ca²⁺]_o to suppress PTH secretion. In one kindred, an Alu-repetitive element is inserted after residue 876 (comparable with 877 in the bovine CaR) in the C-terminal tail. This causes premature termination of the CaR protein. As a result, the ability of the receptor to mobilize intracellular Ca²⁺ is markedly suppressed (to <10% of wt control) (13), despite adequate cell-surface expression. This supports our hypothesis that deleting the putative -helical domain in the region between 877 and 891 interferes with signaling by the CaR.

In another kindred with FHH, a Phe  Leu mutation at residue 881 of the CaR (Phe⁸⁸² in the bovine CaR) causes reduced CaR-dependent mobilization of intracellular Ca²⁺ and shifts the ED₅₀ value of the receptor to the right (15). The properties of this mutant CaR are consistent with our Phe⁸⁸²  Ala CaR mutant. Based on studies with HEK-293 cells, a mutation of Phe⁸⁸² to Ala is predicted to cause intracellular retention of receptor protein and perhaps a reduced complement of signaling (i.e. wt) CaRs on the cell surface. Such intracellular retention of CaRs in parathyroid cells could readily explain a reduced responsiveness of PTH secretion to increased [Ca²⁺]_o in patients.

A family with autosomal dominant hypocalcemia and gain of CaR function (i.e. enhanced suppression of PTH secretion by high [Ca²⁺]_o) was shown to have an in-frame deletion of amino acids Ser⁸⁹⁵ to Val¹⁰⁷⁵. This deletion mutant, expressed in HEK-293 cells, shows increased levels of membrane CaR expression, suggesting a "dominant positive" effect of the loss of this portion of the CaR tail on expression (16). Taken together, available in vitro data on the CaR tail mutants plus information on naturally occurring human CaR mutations in this region underscore the multiple functions served by the CaR tail in extracellular Ca²⁺ sensing in vivo.

	ACKNOWLEDGEMENT

We acknowledge the helpful discussions of Dr. Robert Nissenson in the Endocrine Research Unit of the San Francisco Veterans Affairs Medical Center.

	FOOTNOTES

^* This work was supported by a Department of Veterans Affairs Merit Review and National Institutes of Health Grants DK 43400 and DK 55846.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: Endocrine Research Unit, 111N, San Francisco, Dept. of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2089; Fax: 415-750-6929; E-mail: dolores@itsa.ucsf.edu.

Published, JBC Papers in Press, September 4, 2001, DOI 10.1074/jbc.M104834200

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_o, extracellular [Ca²⁺]; CaR, calcium receptor; PLC, phospholipase C; TA, tandem-Ala; InsP, inositol phosphate; FHH, familial hypocalciuric hypercalcemia; wt, wild type; PNG-F, peptide N-glycosidase F; PCR, polymerase chain reaction; Endo-H, endoglycosidase H; PTH, parathyroid hormone; NSHPT, neonatal severe hyperparathyroidism; ER, endoplasmic reticulum.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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