Identification and Functional Characterization of Novel Calcium ...

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The Journal of Clinical Endocrinology & Metabolism 87(3):1309 –1318 Copyright © 2002 by The Endocrine Society

Identification and Functional Characterization of Novel Calcium-Sensing Receptor Mutations in Familial Hypocalciuric Hypercalcemia and Autosomal Dominant Hypocalcemia LILIA D’SOUZA-LI*, BING YANG, LUCIE CANAFF*, MEI BAI, DAVID A. HANLEY, MURAT BASTEPE, SONIA R. SALISBURY, EDWARD M. BROWN, DAVID E. C. COLE, GEOFFREY N. HENDY

AND

Departments of Medicine, Human Genetics, and Physiology, McGill University and Calcium Research Laboratory, Royal Victoria Hospital (L.D.-L., B.Y., L.C., G.N.H.), Montre´al, Quebe´c, Canada H3A 1A1; Department of Medicine, University of Calgary (D.A.H.), Alberta, Canada T2N 4N1; Department of Medicine, Harvard Medical School and Endocrine Unit, Massachusetts General Hospital (M.B.), Boston, Massachusetts 02114; Departments of Pediatrics and Medicine, Dalhousie University (S.R.S.), Halifax, Nova Scotia, Canada B3J 3G9; Endocrine-Hypertension Division and Membrane Biology Program, Brigham and Women’s Hospital, and Harvard Medical School (M.B., E.M.B.), Boston, Massachusetts 02114; and Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto and The Banting Institute (D.E.C.C.), Toronto, Ontario, Canada M5G 1L5 Familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcemia (ADH), in which calcium homeostasis is disordered, are associated with mutations in the calcium-sensing receptor (CASR). Six unrelated kindreds with FHH and/or NSHPT and two unrelated kindreds with ADH were studied. Direct sequence analysis of the exons of the CASR gene identified heterozygous mutations in six of the kindreds with FHH and in one of those with ADH. We performed functional analyses on the novel missense and insertion/frameshift mutants by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. All mutant receptors were expressed at a similar level to that of the wild type; however, whereas mutants R220W and A835T (the ADH mutant) were fully glycosylated

and were visualized on the cell surface, glycosylation of mutants G549R and C850ˆ851 ins/fs was impaired, resulting in reduced cell surface staining. In fura-2-loaded HEK293 cells expressing the wild-type or mutant receptors, the inactivating R220W mutant produced a significant shift to the right relative to the wild-type CASR in the cytosolic calcium response to increasing extracellular calcium concentrations and the G549R and C850ˆ851 ins/fs mutants were without detectable activity. The activating A835T mutation resulted in a shift to the left in the cytosolic calcium response to extracellular calcium concentrations relative to the wild type. Our studies have identified novel CASR mutations that alter the function of the CASR in several different ways. (J Clin Endocrinol Metab 87: 1309 –1318, 2002)

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lular stores ([Ca2⫹]i), and diacylglycerol formation, which leads to PKC activation. Two disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), can be associated with loss of CASR function. FHH (also known as familial benign hypercalcemia) is an autosomal dominant disorder characterized by modest elevation of the serum calcium concentration that is generally asymptomatic, relative hypocalciuria, and PTH levels that are not suppressed by the hypercalcemia and are inappropriately normal (4, 5). Although the inheritance of a single copy of a mutated CASR gene causes FHH, homozygous individuals who inherit two inactive gene copies may have NSHPT, which is characterized by marked hypercalcemia, skeletal demineralization, and parathyroid hyperplasia and, without parathyroidectomy, can be fatal (6 – 8). Several different inactivating mutations of the CASR gene have been found in patients with FHH or NSHPT (9 –12). These are predominantly missense mutations that are clustered in the proximal portion of the extracellular domain, which may constitute the calcium-binding site, and in the transmembrane domain (13, 14).

HE HUMAN CALCIUM-SENSING receptor (CASR), encoded by six exons of the CASR gene located on chromosome 3q13.3–21 (1), is a 1078-amino acid glycoprotein with a predicted topology of a large extracellular domain, a seven-transmembrane-spanning region, and an intracellular tail (2, 3). This G protein-coupled receptor is expressed abundantly in the parathyroid and, to a lesser extent, along the length of the kidney tubule. The CASR plays a critical role in calcium homeostasis by sensing elevations in the extracellular calcium concentration ([Ca2⫹]o), leading to inhibition of PTH secretion and renal calcium reabsorption. Activation of the CASR can couple the [Ca2⫹]o signal to several different intracellular effectors. The best characterized pathway involves G␣q/11, which activates PLC. This results in IP3 generation, which causes the release of calcium from intracelAbbreviations: ADH, Autosomal dominant hypocalcemia; [Ca2⫹]i, cytosolic calcium; [Ca2⫹]o, extracellular calcium concentration; CASR, calcium-sensing receptor; Endo H, endoglycosidase H; FHH, familial hypocalciuric hypercalcemic; NSHPT, neonatal hyperparathyroidism; PNGase F, peptide-N-glycosidase F.

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J Clin Endocrinol Metab, March 2002, 87(3):1309 –1318

Another disorder, autosomal dominant hypocalcemia (ADH), can be associated with activating mutations in the CASR. Affected ADH individuals have inappropriately low circulating PTH levels with varying degrees of severity of hypocalcemia and relatively few symptoms. Seizures can occur, although parathesias, tetany, and laryngospasm are uncommon. Several different CASR activating mutations have been identified in patients with ADH (15–18). These are clustered in two parts of the receptor, the proximal extracellular domain and the transmembrane region (14). The aims of the present study were to identify novel mutations in the CASR gene in FHH/NSHPT and ADH kindreds, and to examine the effects of these mutations on the expression of the CASR protein, its maturation and trafficking to the plasma membrane, and its ability to couple changes in [Ca2⫹]o to intracellular signaling pathways. Subjects and Methods Families Consent was obtained from all participants or their guardians in accordance with local institutional guidelines. Venous blood samples were collected from affected and unaffected members of FHH and ADH families (Fig. 1). Some members of family C have been described previously (19). Family E is unusual in that two of the affected members have relative hypercalciuria rather than hypocalciuria (20). In this kindred the disease locus was shown to segregate with chromosome 3q markers (21). Clinical details of the probands are summarized in Table 1.

Sequence analysis of the CASR gene Leukocyte DNA was isolated using standard methods. Nine primer pairs were used to amplify exons 2–7 of the CASR gene, which encode the CASR protein. Forward and reverse primers were modified at their 5⬘-ends by addition of T7 and T3 promoter sequences to aid in the subsequent direct sequencing of gel-purified products. Primers 2F/2R, 3F, 4AF/ 4AR, 4BF/4BR, 5F/5R, 6F/6R, 7CR, and 7DR were described by Pollak et al. (9), and primer 7GF was described by Pearce et al., (12). Novel primers are: 3R⬘, 5⬘-ACCCAAGCCTCGATCTCTGATCCTAG-3⬘; 7BF (to match 7CR), 5⬘-TCAAGCTACCGCAACCAGGAGCTGGA-3⬘; 7ER (to match 7GF), 5⬘-AGTCTGTGCCACACAATAACTCACTCT-3⬘; and 7DF (to match 7DR), 5⬘-TGCCGCTGCCAAAGATGACCTTCTG-3⬘. Amplification reactions were conducted as previously described (11), and after gel purification PCR products were directly sequenced using a 373A automated sequenator (PE Applied Biosystems, Foster City, CA) located at the Sheldon Biotechnology Center of McGill University.

Restriction enzyme analysis Cosegregation of the DNA sequence abnormality in affected family members was confirmed by digesting PCR fragments with specific restriction enzymes. When the mutation did not alter a naturally occurring site, a mutant primer was constructed to introduce a base pair change adjacent to the mutation, thereby creating a specific restriction enzyme site not present in the wild-type sequence.

c-Myc-tagged CASR cDNA A modified CASR cDNA was constructed encoding a c-Myc epitope tag inserted between amino acids 22 and 23 just after the signal peptide cleavage site at amino acid 19 in the CASR NH2-terminal region. This was achieved by performing two PCRs using the high fidelity Pfu polymerase enzyme and plasmid HuPCaR4.0 (provided by Dr. J. E. Garrett) as template. For the first PCR the forward primer was 5⬘-TAGAAGCTTCATCCCTTGCCCTGGAGAGACGGCAGA-3⬘ in which the underlined sequence is a HindIII restriction enzyme site, followed by ⫺30 to ⫺4 of the CASR cDNA (where ⫹1 represents the A of the ATG initiation codon). The reverse primer was 5⬘-TGAGTCGACCAGATCCTCTTCTGAAATCAGTTTT-3⬘ in which the underlined sequence is a SalI restric-

D’Souza-Li et al. • Mutations in the CASR Gene

tion enzyme site, and the sequence in bold is the c-Myc epitope followed by nucleotides complementary to the codons for amino acids 15–22 of the CASR. The PCR product of 144 bp was treated with Taq polymerase to add A to the 3⬘-ends and cloned into a TA vector. For the second PCR the forward primer was 5⬘- CAAGTCGACGACCAGCGAGCCCAAAAGAAGGGG-3⬘ in which the underlined sequence is a SalI restriction enzyme site, followed by a sequence encoding CASR amino acids 23–30. The reverse primer was 5⬘-GAATTCCCGGAAGCCTGGGATCTG-3⬘, which is complementary to the codons for amino acids 326 –333. The PCR product of 932 bp was cloned into a TA vector. The vector carrying the first PCR product was opened by digestion with SalI and EcoRV (which creates a blunt end), and the second PCR product was excised with SalI and MlsI (which creates a blunt end) and, after gel purification, was cloned downstream of the first PCR product. From this clone the HindIII-EagI insert was ligated into a HindIII-EagIdigested CASR cDNA in a pcDNAIAmp vector (plasmid HuPCaR4.0). The entire c-Myc-tagged cDNA was excised with HindIII and XbaI and inserted into pcDNA3 to be used as a template for site-directed mutagenesis and subsequent transfection studies. Throughout the procedure the cloned PCR products and inserts of the final construct were sequenced to confirm their correctness.

Site-directed mutagenesis The Quik Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used. For each mutation, the primers were complementary with the mutant sequence placed in the middle. The primers were annealed to the template (either an untagged or c-Myc-tagged human CASR cDNA in pcDNA3), and 12 rounds of extension were performed with Pfu Turbo DNA polymerase, followed by digestion of the template with DpnI enzyme. The reaction was used to transform an Escherichia coli strain (XLI-Blue) that can incorporate nicked DNA and repair it, and colonies were screened by restriction enzyme digestion for the presence of the mutation. The correctness of all constructs was confirmed by sequencing.

Transient transfection of human CASR cDNA Human embryonic kidney (HEK293) cells (provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) were cultured and transfected with the human CASR cDNAs as previously described (22). Forty-eight hours after transfection, cells were harvested for total cellular protein extraction (23) or crude plasma membrane preparation (22) as previously described. The N-linked glycosylation status of the recombinant receptors was evaluated in crude plasma membranes by digestion with endoglycosidase H (Endo H) and peptide-N-glycosidase F (PNGase F) as previously described (24). Western blot analysis of either total cell extracts (23) or crude plasma membranes (22) was performed. The primary antibodies used were AS2011, an affinity-purified rabbit polyclonal antibody raised against a peptide comprising CASR amino acids 215–235 coupled to keyhole limpet hemocyanin, and the c-Myc 9E10 monoclonal antibody isolated from mouse ascites fluid.

Fluorescence immunocytochemistry and confocal microscopy HEK293 cells were transiently transfected with either c-Myc-tagged wild-type or mutant CASR cDNA. Forty-eight hours after transfection, the PBS-washed cells were fixed in 4% paraformaldehyde/0.12 m sucrose. Cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min if required. Washed cells were incubated in 6% goat serum for 1 h and then at 4 C overnight with 9E10 c-Myc monoclonal antibody at a 1:1000 dilution. For permeabilized cells, 0.1% Triton X-100 was added to all solutions. Washed cells were incubated for 1 h with a goat antimouse Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Slides were mounted with Prolong mount medium (Molecular Probes, Inc., Eugene, OR), dried, and visualized by confocal microscopy. The Carl Zeiss LSM 410 laser scanning microscope (New York, NY) was equipped with a 568-nm excitation wavelength and a long-pass 590-nm emission filter. A pinhole size of 16 was employed, and the same zoom and contrast/brightness settings were used for each image.


J Clin Endocrinol Metab, March 2002, 87(3):1309 –1318 1311

FIG. 1. Pedigrees of families with FHH (A–F) and ADH (G and H). Clinical status is indicated by open symbols (unaffected) and solid symbols (affected). Individuals with normal results on biochemical assessment are shown by a quartered symbol. The clinical status of individual III5 of family D is not known (?). Probands are indicated by an arrow: the biochemical details of each proband are given in Table 1. The presence (⫹) or absence (⫺) of a mutation in tested family members is shown.

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TABLE 1. Biochemical characteristics of FHH and ADH probands Family

Serum total calcium (mmol/liter)

Proband

FHH families A III-1 B III-6 C III-8 D III-7 E II-3 F I-2 ADH families G III-8 H III-4

2.83 2.9 (2.2–2.65)b 2.8 (2.1–2.55) 2.86 3.00 (2.2–2.62) 2.83 (2.2–2.6)

Serum magnesium (mmol/liter)

Blood ionized calcium (mmol/liter)

Serum phosphate (mmol/liter)

Serum creatinine (␮mol/liter)

1.43 1.56 (1.15–1.28) ND 1.59 ND 1.46 (1.17–1.33)

1.3 0.83 0.67 (0.8 –1.5) 0.91 (0.81–1.3) 0.99 (0.8 –1.5) 1.01 (0.90 –1.45)

103 98 93 (60 –120) 95 71 68 (50 –110)

ND ND ND 1.4 (0.4 –1.3 ng/ml) 1.28 (0.6 –1.0) 110 (13–54 ng/liter) ND 2.5 (1.3–5.7 pM) 0.92 (0.6 –1.0) 96 (⬍100) ND 5.0 (1.0 – 6.5 pM)

0.004 0.005 0.012 0.015 0.025 (⬎0.016) 0.009 (⬎0.016)

1.19 2.58

100 97

0.63

0.088 0.015

1.64 1.7

ND ND

Plasma PTH

3 (10 – 60 ng/liter) 8

ND

Urinary Ca/Cr clearance ratioa

ND, Not determined. UCa ⫻ SCr/UCr ⫻ SCa) where UCa, UCr, SCa and SCr are the 24-h urinary excretions of calcium and creatinine and the serum total calcium and creatinine concentrations, respectively. b Where known, normal ranges are given in parentheses. a

TABLE 2. Summary of CASR mutations in FHH and ADH kindreds Location

Systematic name

Trivial name

RE

FHH kindred A B C D E F

E2-ECD E3-ECD E4-ECD E6-ECD E7-ICL3 E7-TM7

c.19ˆ20.insT c.413.C⬎T c.658.C⬎T c.1645.G⬎A c.2383.C⬎T c.2550ˆ2551. insCCAG

C7fs-2X47 T138M R220W G549R R795W C850ˆ851 Pvfs-2X981

Seq lose AccIa lose Eag1 lose BsmFI lose NciI gain NarI

ADH kindred G H

E7-ECL No mutation found

c.2503.G⬎A

A835T

gain BglIa

RE, Restriction enzyme; Seq, all affecteds sequenced; ECD, extracellular domain; ICL, intracellular loop; TM, transmembrane domain; ECL, extracellular loop. a Mutant primer.

Measurement of Ca2⫹i by fluorometry in cell populations Coverslips coated with HEK293 cells that had been transfected with wild-type or mutant CASR cDNAs were loaded with fura-2/AM, and Ca2⫹i was measured as previously described (24).

Statistics For the fura-2 experiments, comparison of the EC50 values was performed as previously described (24) using ANOVA or Duncan’s multiple comparison test (25). P ⬍ 0.05 was taken to indicate a statistically significant difference.

Results Mutations in CASR

Direct sequence analysis of PCR-amplified CASR exons identified heterozygous mutations in the probands of seven families, six with FHH and one with ADH (Table 2). Of these, four were novel, and three FHH mutations had been reported previously in apparently unrelated families (9, 26, 27). In one of the previously reported cases, this was a change of C to T at bp 413 in exon 3, altering codon 138 from threonine to methionine (family B). This did not alter a restriction site, so a mutant primer was constructed introducing an AccI site into the wild-type exon 3 product that was disrupted by the mutation. In the proband of family C, a mutation of a C to T at bp 658 in exon 4 changed codon 220 from arginine to tryptophan. This mutation was confirmed to segregate with other affected family C members by restriction enzyme anal-

ysis as it disrupts an EagI site. In the third case there was a change of C to T at bp 2383 in exon 7, altering codon 795 from arginine to tryptophan, which disrupted an NciI site (family E). Of the three novel FHH mutations, one was missense, and two were of the insertion type. In family A there was an insertion of a T between bp 19 and 20 in exon 2 at codon 7 at the beginning of the protein within the putative signal sequence (Fig. 2A). This results in a change of cysteine to leucine and a frameshift, creating a stop codon at position 47. CASR exon 2 was PCR-amplified and subcloned, and both wild-type and mutant alleles were sequenced from the affected family members (Fig. 2A). In family D a G to A change at bp 1645 caused a substitution of glycine by arginine at codon 549 and disrupted a BsmFI restriction site. In family F four additional nucleotides (CCAG) were inserted between bp 2550 and 2551 encoding the seventh transmembrane domain in exon 7. This resulted in insertion of a proline after codon 850 and a frameshift creating a stop codon 393 bp downstream. In the proband from family G with ADH a G was substituted by an A at bp 2503 changing amino acid 835 from alanine to threonine (Fig. 2B). As this change did not alter a restriction site, a mutant primer was designed that created a BglI site in the wild-type exon 7 PCR product that is disrupted by the mutation. By restriction enzyme analysis it was demonstrated that the mutation segregated with affected family members (Fig. 2B).



FIG. 2. Detection of mutations in the CASR gene. A, Left panel, Direct sequence analysis of genomic DNA (sense strand shown) of patient III-1 of family A revealed a heterozygous insertion mutation that was confirmed (right panel) by sequencing individual subclones (antisense strand shown) of the PCR product. B, Left panel, Direct sequence analysis of genomic DNA of patient III-8 of family G revealed a heterozygous missense mutation. Right panel, This mutation resulted in a gain of a BglI site when amplified with a modified primer (see text), facilitating its detection in other family members.

Expression of wild-type and mutant CASRs in HEK293 cells

By site-directed mutagenesis c-Myc-tagged wild-type and mutant CASR cDNAs were prepared and were transiently transfected into HEK293 cells. Western blot analysis of total cell extracts used either a polyclonal anti-CASR antibody (Fig. 3A) or an antibody to the c-Myc epitope tag (Fig. 3B). All mutant receptors were expressed at approximately equivalent levels to that of the wild-type receptor. All were well recognized by both antibodies, with the exception of the R220W mutant, which was only detected with the c-Myc antibody. This is unsurprising given the fact that the CASR antibody was raised against a peptide corresponding to CASR amino acids 215–235. This result helps to place the epitope recognized by this antibody as being toward the NH2-terminus of this sequence. The CASR exists in both monomeric and dimeric forms: the monomeric unglycosylated species is 120 kDa, the core glycosylated (immature) species is 140 kDa, and the mature, fully glycosylated species is 160 kDa (24). The predominant monomeric species observed in the total cell extracts was the 140-kDa form, with lesser amounts of the 160-kDa form being observed not only in wild-type, but also in mutant R220W- and A835T-trans-

fected cells. In the case of the C850ˆ851 ins/fs mutant, the monomeric species had a gel mobility consistent with it being some 10 kDa smaller than the wild type. Little of the mature glycosylated form was apparent for the G549R and C850ˆ851 ins/fs mutant. Higher molecular mass forms, presumably dimers, were seen for all receptors, wild type and mutants. Glycosylation status of the wild-type and mutant receptors

The CASR undergoes core (immature) N-linked glycosylation in the endoplasmic reticulum. Once appropriately folded, the core-glycosylated receptor transits through the Golgi apparatus, becomes fully (mature) glycosylated, and is expressed at the cell surface. If the receptor is misfolded, it is retained within the endoplasmic reticulum in the immature glycosylated form and is not trafficked to the plasma membrane. To confirm the identification of nonglycosylated and glycosylated forms of the CASR as described in the previous section and to explore in further detail the maturation status of the mutant receptors, enzymatic deglycosylation experiments were performed on crude plasma membranes of CASR cDNA-transfected HEK293 cells. Asparagine-linked glycosyl moieties can be enzymatically removed by PNGase F and

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FIG. 3. Western blot analysis of total cell extracts of HEK293 cells transiently transfected with either wild-type or mutant c-Myc-tagged CASR cDNAs. A, Recombinant proteins stained with a polyclonal antibody raised against CASR amino acids 215–235. B, Recombinant proteins stained with c-Myc monoclonal antibody 9E10.

FIG. 4. Enzymatic deglycosylation analysis of crude plasma membrane preparations isolated from HEK293 cells transiently transfected with either wild-type or mutant c-Myc-tagged CASR cDNAs. The preparations were either mock-treated (no treatment) or treated with Endo H or PNGase F and subjected to SDS-PAGE. The blot was stained with c-Myc monoclonal antibody 9E10.


Endo H. Whereas PNGase F can cleave all forms (mature and immature) of N-linked sugar chains, Endo H can only cleave the core-linked immature sugar chains and is inactive once a glycoprotein has been modified by mannosiadase II, an enzyme present in the cis-Golgi. Western blot analysis of crude plasma membranes from cells transfected with wildtype CASR (c-Myc-tagged) cDNA showed that the 160- and 140-kDa species were sensitive to PNGase F. In contrast, only the 140-kDa species was sensitive to Endo H, confirming that the 160-kDa species is the fully glycosylated form, and the 140-kDa species is the immature core-glycosylated form (Fig. 4). Plasma membranes from wild-type, R220W, and A835T mutant transfected cells demonstrated approximately equivalent amounts of the mature and immature forms, whereas for all other mutants there was relatively less of the mature form. This was most marked for G549R and C850ˆ851 ins/fs. For the latter truncation mutant, the core-glycosylated form was slightly less than 120 kDa, and treatment with PNGase F and Endo H further reduced this in size. In all cases wildtype and mutants, whereas treatment with PNGase F reduced the majority of the immunostained material to a (nonglycosylated) species of 120 kDa, some slightly higher molecular mass material was also evident. This has been observed previously for the wild-type receptor (24) and indicates that the mature receptor may have other posttranslational modifications in addition to the N-linked glycosylation.


Fluorescence immunocytochemistry and confocal microscopy

To analyze whether the CASR mutants were expressed on the cell surface, fluorescence immunocytochemistry was performed on HEK293 cells transiently transfected with c-Myctagged wild-type and mutant CASR cDNAs. The analysis was performed in 1) nonpermeabilized cells to detect cell surface staining only (indicating either appropriate receptor maturation and trafficking to the plasma membrane or, in the case of absent staining, misfolding and trapping within the cell), and 2) in permeabilized cells to assess the amount of receptor normally present intracellularly and undergoing maturation and trafficking to the plasma membrane or the amount of a mutant receptor that had become trapped within the endoplasmic reticulum. Cells mock-transfected or transfected with untagged re-

FIG. 5. Fluorescence immunocytochemistry and confocal microscopy. Immunostaining was performed with c-Myc monoclonal antibody 9E10, and detection was made using goat antimouse Cy3-conjugated secondary antibody. A, Examples of fields of nonpermeabilized and permeabilized HEK293 cells transfected with either wild-type or mutant c-Myc-tagged CASR receptor cDNA. B, Examples of individual permeabilized HEK293 cells transfected with either wild-type or mutant c-Myctagged CASR receptor cDNA. The percentage of positively stained HEK293 cells transiently transfected with either wild-type or mutant c-Myc-tagged CASR cDNAs: C, nonpermeabilized cells; D, permeabilized cells. *, P ⬍ 0.01 compared with wild type.


ceptor CASR DNA showed no specific staining with the c-Myc antibody (Fig. 5, A and B, and data not shown). Strong staining was observed at the cell surface of nonpermeabilized HEK293 cells that had been transfected with the c-Myctagged, wild-type receptor. Permeabilization of such cells revealed further intracellular perinuclear staining associated with the endoplasmic reticulum and Golgi apparatus (Fig. 5B). No nuclear staining was observed. Nonpermeabilized and permeabilized cells that had been transfected with either the R220W inactivating mutant or the A835T activating mutant showed a similar pattern of staining to that of the wild type. For the G549R and C850ˆ851 ins/fs mutants far fewer nonpermeabilized cells stained relative to permeabilized cells (Fig. 5A). Relative numbers were quantitated for all constructs by counting stained cells within a given field area

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(Fig. 5, C and D). Thus, although the numbers of nonpermeabilized and permeabilized cells were equivalent for the wild-type and A835T mutant, slightly fewer nonpermeabilized cells were observed for the R220W mutant, and much less (10 –15%) were apparent for the G549R and C850ˆ851 ins/fs mutants. Generally, there was good agreement between the relative amounts of the Endo H-resistant mature species vs. Endo H-sensitive core-glycosylated species, as demonstrated by Western analysis of crude plasma membrane (Fig. 4) and the amount of cell surface expression vs. intracellular immunofluorescent staining (Fig. 5). [Ca2⫹]i responses of CASR mutants to Ca[2⫹]o

The ability of the mutant receptors relative to wild-type receptor to respond to [Ca2⫹]o and mediate increases in Ca2⫹i was examined in fura-2-loaded cells using dual wave-length fluorometry. The c-Myc-tagged wild-type CASR cDNA when transiently expressed in HEK293 cells showed a halfmaximal response (EC50) of 4.0 ⫾ 0.1 mm (mean ⫹ se), similar to the EC50 value of 3.8 ⫾ 0.1 mm for the untagged-wild-type receptor (Fig. 6A). This together with the finding that the levels of protein expression for the two receptors determined by immunoblot of total cell extract as well as crude plasma membrane preparations and staining with the anti-CASR polyclonal antibody were similar provided reassurance that insertion of the c-Myc epitope tag had no adverse effect on the expression or function of the receptor. The R220W mutant had a dose-response curve shifted to the right relative to that of wild-type with an EC50 value of 15.4 ⫾ 0.5 mm (Fig. 6B); however, at very high [Ca2⫹]o the same maximal Ca2⫹i response was achieved as with wild-type. The G549R and C850ˆ851 ins/fs mutants were completely unresponsive to [Ca2⫹]o up to 50 mm. In contrast, the activating mutant A835T, although starting from the same baseline level as the wild type, showed a significant leftward shift in its doseresponse curve with an EC50 of 2.86 ⫾ 0.02 mm (Fig. 6C) and a maximum response that was the same as the wild-type receptor. Discussion

CASR mutations were identified in all six FHH/NSHPT kindreds examined: three were novel and three had been described in other families previously. In one ADH family a novel mutation was identified, and in a second kindred no mutation was found. The novel mutations (with the exception of C7fs-2X47) were engineered into a human CASR cDNA, and their effects on receptor expression, glycosylation, appearance at the plasma membrane, and cell signaling were assessed after transient transfection into HEK293 cells. Western blot analysis of cells transfected with the wild-type CASR cDNA showed several immunoreactive species of differing mobilities. These are the nonglycosylated (120 kDa), immature mannose-rich glycosylated (140 kDa), and mature glycosylated (160 kDa) forms of the CASR as well as higher molecular mass, presumably dimeric forms (⬎240 kDa), as described previously (24, 28). The mutants were expressed at levels comparable to the wild-type, although for two of them, G549R and C850ˆ851PVfs-2x98, a complete lack of the mature glycosylated form, which is normally a less abundant species

FIG. 6. [Ca2⫹]o-evoked increases in [Ca2⫹]i in fura-2-loaded HEK293 cells transiently transfected with either wild-type or mutant CASR cDNAs. A, c-Myc-tagged and untagged wild-type CASR. B, c-Myctagged wild-type and inactivating mutant CASRs. C, c-Myc-tagged wild-type and activating mutant CASRs. Values shown are the mean ⫾ SEM of 10 estimations.

in the whole cell extract, could be noted indicating an abnormality in processing of the receptor. The abilities of the mutant CASRs to be properly glycosylated and trafficked to the cell surface were examined in experiments using enzy-



matic deglycosylation of crude plasma membranes of the transfected cells. For the R220W and A835T mutants, mature glycosylated receptor was present; in contrast, there was little of this form for the G549R and C850ˆ851fs mutants, indicating that they were not properly glycosylated and were retained in the endoplasmic reticulum or early Golgi complex. This was confirmed by the immunofluorescence studies, which showed that the R220W and A835T mutants were expressed at the cell surface at levels comparable to that of the wild-type, whereas the G549R and C850ˆ851fs mutants were poorly detected at the cell surface in the nonpermeabilized cells. The mutants showed similar levels to wildtype in permeabilized cells, but although R220W and A835T, like the wild type, demonstrated strong staining at the cell surface, the G549R and C850ˆ851fs mutants did not, suggesting retention intracellularly. The R220W mutation has recently been described (27) in an unrelated FHH family, and mutation to a different amino acid at the same codon, R220Q, has been reported (29), although functional studies were not carried out in either case. The majority of the inactivating mutations lie in the NH2-terminal third of the CASR’s extracellular domain, with one grouping of seven missense mutations including R220W spanning amino acids 215–227, suggesting the importance of this region for ligand-receptor interactions. Mutants T138M and R795W, which were studied previously (24), like R220W, achieve mature glycosylation and hence are likely to be expressed at the cell surface, but demonstrate a rightward shift in their relationships between [Ca2⫹]o and intracellular Ca2⫹ signaling (Table 3). However, whereas T138M, like R220W, has the same maximal response as the wild type, the R795W mutant achieves only approximately 10% of this response. The two mutants, G549R and C850ˆ851fs, that were not expressed on the cell surface were completely unable to elevate [Ca2⫹]i at any [Ca2⫹]o. Although the R220W mutant, which was processed properly and expressed at the cell surface, had the same maximal response as the wild-type receptor, the curve was shifted to the right, indicating that this residue is critical for proper ligand-receptor interaction. The R795W mutation is in intracellular loop 3, which is likely to contact G proteins important for CASR signaling. In a previous study, engineered mutants involving other amino acids in intracellular loop 3 were, like R795W, defective in TABLE 3. Functional properties of wild-type and mutant CASRs Construct

Protein expressed

Mature glycosylation

Cell surface expression

Ca2⫹i signalling EC50 [Ca2⫹]0, mM

Wild-type C7fs-2X47 T138Mc R220W G549R R795Wb A835T C850ˆ851

⫹ ND ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ND ⫹ ⫹ ⫺ ⫹ ⫹ ⫺

⫹ ND ND ⫹ ⫺ ND ⫹ ⫺

4.0 ⫾ 0.1a ND 12.4 ⫾ 0.4* 15 ⫾ 0.5* NR 12.8 ⫾ 0.3* 2.86 ⫾ 0.2* NR

ND, Not determined; NR, no response. a Values are means ⫾ SE; values marked with asterisks are significantly (P ⬍ 0.05) different from the wild type. b Data taken from Bai et al. (1996).

Ca2⫹i signaling (30), emphasizing the importance of this loop in mediating efficient coupling to PLC. The A835T mutant was appropriately processed and expressed at the cell surface; however, the EC50 [Ca2⫹]o for Ca2⫹i signaling was significantly shifted to the left. Like most other activating CASR mutations there was no increase in [Ca2⫹]i at low levels of [Ca2⫹]o, and the maximal response to [Ca2⫹]o was equivalent to that of wild-type. It has been postulated that ligand binding to the NH2-terminal part of the extracellular domain of the CASR directly or indirectly modifies the conformation of the transmembrane domain, leading to receptor activation. The A835T mutation lies within extracellular loop 3, suggesting that this loop may participate in this process. Whether the ligand-bound extracellular domain directly contacts the extracellular loops or transmembrane domain remains to be determined. In the majority of FHH cases studied here, the CASR mutations do not predict any phenotype other than mild hypercalcemia and relative hypocalciuria. In one family (kindred E), an R795W mutation that had been identified in an unrelated family (9) was found. This mutant receptor was demonstrated to function in a dominant-negative manner when cotransfected into cells with the wild-type receptor (24). Consistent with this, the serum calcium levels in affected members of kindred E were 3.0 mmol/liter or more, slightly higher than in typical FHH, and in two family members, hypercalciuria, rather than hypocalciuria, was noted (20). In the present study, in all FHH/NSHPT kindreds examined CASR mutations were identified, although in only two thirds of such kindreds have CASR mutations been identified overall, and thus in one third of cases the molecular etiology is unclear. In these cases the disorder could be due to mutations in portions of the CASR gene, 5⬘-regulating regions or introns, yet to be examined. In rare cases a gene other than CASR could be involved. Additionally, the biochemical picture of FHH can occur in patients with anti-CASR autoantibodies and associated autoimmune disorders such as sprue or autoimmune thyroid disease (31). Of the two ADH kindreds examined here, for only one was an activating CASR mutation identified. In the other case reasons similar to those outlined above for failure to identify a mutation in an FHH kindred could also apply. Antibodies against the CASR have been found in patients with type 1 autoimmune polyglandular syndrome or acquired hypoparathyroidism associated with autoimmune hypothyroidism (32), although patients in kindred H did not have the findings that accompany these autoimmune disorders. The functional analysis of the mutant CASRs provided a full explanation of the clinical phenotype in the patients. In no case was the amount of expression reduced or was the receptor’s ability to form dimers compromised. However, functional alterations, such as impaired protein maturation and lack of cell surface expression, reduced (apparent) ligand binding, or altered receptor coupling to cellular signaling pathways contributed to one or the other mutant receptor’s altered activity. Continued identification and characterization of naturally occurring CASR mutations will be critical for furthering our knowledge of the precise workings of this important receptor.

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Acknowledgments We thank all family members for their participation and the following for contributing laboratory and clinical data and patient samples: Drs. F. Richard Bringhurst, John D. Crawford, Sudip Datta, Lois Donovan, Daniel Drucker, Paul Goodyer, Natasa Janicic, Harald Ju¨ ppner, Sang Whay Kooh, Robb M. Meyer, Zdenka Pausova, and Rima Rozen. Drs. Philip Barker and Janet Henderson provided the c-Myc monoclonal antibody, and Drs. J. E. Garrett and K. V. Rogers provided plasmid pHuPCaR4.0 and HEK293 cells. Received September 21, 2001. Accepted November 13, 2001. Address all correspondence and requests for reprints to: Geoffrey N. Hendy, Ph.D., Calcium Research Laboratory, Room H4.67, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebe´ c, Canada H3A 1A1. E-mail: [email protected]. This work was supported by research grants from the Canadian Institutes of Health Research (MT-9315) and the Kidney Foundation of Canada (to G.N.H.), NIH Grants (DK-48330 and DK-41415), and The St. Giles Foundation (to E.M.B.). * Recipient of a doctoral fellowship from the Canadian Institutes of Health Research.

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