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cek and
Geoffrey N. Hendy
Departments of Nephrology, Endocrinology, and Metabolic Diseases (A.Wy., J.C., F.K., A.Wi.), Medical University of Silesia, Katowice, 40-055 Poland; and Departments of Medicine, Human Genetics, and Physiology (S.P., L.C., G.N.H.), McGill University and Calcium Research Laboratory, Royal Victoria Hospital, Montréal, Québec, H3A 1A1 Canada
Address all correspondence and requests for reprints to: Dr. Geoffrey N. Hendy, Calcium Research Laboratory, Royal Victoria Hospital, Room H4.67, 687 Pine Avenue West, Montreal, Quebec H3A 1A1 Canada. E-mail: geoffrey.hendy{at}mcgill.ca.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The FBHH trait was mapped to chromosome 3q by linkage analysis (17), and the gene encoding the calcium-sensing receptor (CaSR) was later mapped to this locus by fluorescence in situ hybridization (18). The CaSR is a cell-surface G protein-coupled receptor expressed in parathyroid chief cells and renal tubule cells (19). Binding of calcium to the extracellular domain of the CaSR activates the receptor and intracellular signaling pathways that inhibit PTH secretion and urinary calcium (and magnesium) reabsorption (20). Different inactivating missense mutations in the CASRgene were identified in affected members of three unrelated FBHH families in 1993 (21). Since that time, approximately 50 unique inactivating mutations have been reported with several being recurrent (22, 23, 24, 25). Missense mutations are by far the most common class identified, with some nonsense mutations, and Alu element insertion (26) and splice-site (27) mutations have also been characterized.
In this report, we present a Polish family with FBHH resulting from an inactivating CASR germline mutation. The mutation was engineered into a CaSR cDNA expression vector and functional characterization of the mutant made with respect to expression, both intracellularly and at the plasma membrane in transfected kidney cells, and its ability to activate a cell-signaling pathway, all relative to the wild-type receptor. In addition, a functional comparison was made with a CaSR harboring a mutation at the same codon but to a different amino acid, previously identified and presenting as neonatal hyperparathyroidism.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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), a 60-yr-old woman with moderate hypercalcemia, came to clinical attention because of suspected primary hyperparathyroidism. She complained of nonspecific bone pain, arthralgia, hand paresthesias, muscle cramps, soreness of the eyes, and insomnia. Her blood pressure was normal (130/70 mm Hg). Serum total and ionized calcium concentrations were elevated [11.2 mg/dl (2.8 mmol/liter) and 6.24 mg/dl (1.56 mmol/liter), respectively], but serum phosphate [3.38 mg/dl (1.09 mmol/liter)] and PTH (46 pg/ml) levels were normal. Serum alkaline phosphatase activity (90 IU/liter), serum calcitonin (48 pg/ml), 25-hydroxyvitamin D3 (42 ng/ml), and creatinine (60 µmol/liter) levels were normal. Skeletal x-ray showed focal decalcification of wrist bones and small periarticular cysts and erosions of finger phalanges, without decreased total bone mineral density (1.008 g/cm2; Z score = –0.07). There was no sign or symptom of kidney stones or nephrocalcinosis, and urinary calcium excretion was not measured at this time. Ultrasound examination of the neck revealed a hypoechogenic structure 5 mm in diameter behind the inferior pole of the left thyroid gland. The diagnosis of primary hyperparathyroidism was made and the patient referred for surgery.
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After the unsuccessful parathyroidectomy, serum chemistries were repeated and the urinary calcium excretion was measured (Table 1, I-1). The low calcium-to-creatinine clearance ratio led to a diagnosis of benign hypocalciuric hypercalcemia. The screening of additional family members was undertaken (Fig. 1
and Table 1). The characteristic pattern of hypercalcemia, inappropriately normal serum PTH, and low urinary calcium-creatinine clearance ratio was observed in the patient’s daughters (II-1 and II-3, Fig. 1) and granddaughter (III-1, Fig. 1). Her grandson (III-2, Fig. 1) was normal. All subjects gave informed consent, and the study was conducted according to the Helsinki Declaration and was approved by the ethical committees of the Medical University of Silesia and the Royal Victoria Hospital.
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Leukocyte DNA was isolated using standard methods. Exons 2–7 of the CASR were amplified as described (28). Gel purified PCR products were directly sequenced. For all family members, CASR exon 4 was amplified and digested with Hpy188I to test for the presence of the mutation.
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 (c-Myc-tagged human CaSR cDNA in pcDNA3.1), 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 (29). Forty-eight hours after transfection, cells were harvested for total cellular protein extraction. Western blot analysis of total cell extracts was performed. The primary antibody used was the c-Myc 9E10 monoclonal antibody.
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. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min if required. Washed cells were incubated in 10% goat serum for 1 h and then incubated with 9E10 c-Myc mouse monoclonal antibody at a 1:100 dilution for 3 h at room temperature. Washed cells were incubated for 1 h with a goat antimouse fluorescein isothiocyanate-conjugated antibody (Molecular Probes, Inc., Eugene, OR). Slides were mounted with mount medium, dried overnight at room temperature, and visualized by confocal microscopy.
MAPK assay
MAPK assay was done as described (30). In brief, a trans-reporting system (Stratagene) was used to measure the activity of Elk-1, an ETS domain transcription factor targeted by MAPK pathways. HEK293 cells were transiently cotransfected with vectors expressing wild-type (0.5 µg) or mutant receptor (0.5 µg) or wild-type and mutant receptor (0.25 µg of each) plus Elk-1 reporter constructs. The next day, cells were serum starved in DMEM containing 0.5 mM CaCl2 for 8 h and cultured in various concentrations of CaCl2 ranging from 0.25–15 mM for 16 h. The cells were washed in PBS and lysed in lysis buffer on ice. Luciferase activity was measured using 45 µl cell lysate and D-luciferin using Fluostar Optima (BMG Labtech GmbH, Offenburg, Germany). Luciferase activity was normalized to ß-galactosidase.
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Results |
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Direct sequence analysis of PCR-amplified CASR exons identified a heterozygous mutation (R227Q, CGA
CAA) in the CaSR extracellular domain encoded by exon 4 of the gene. The mutation led to loss of an Hpy188I site and this provided a convenient diagnostic test to confirm the presence of the mutation in the proband, I-1, and identify the presence (in II-1, II-3, and III-1) and absence (III-2) in other family members (see Figs. 1 and 2, and data not shown).
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By site-directed mutagenesis, c-Myc-tagged R227Q and R227L mutants were created and were transiently transfected into HEK293 cells. Cells were also transfected with either the c-Myc-tagged wild-type (positive control) or empty vector (negative control). Western blot analysis was conducted with an antibody to the c-Myc epitope tag. The two mutant receptors were expressed at equivalent levels to that of the wild-type receptor. 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 (31). The predominant monomeric species observed (see Fig. 3) was the 140-kDa form with the 160-kDa form present (but in lesser amounts) in wild-type- but also the mutant R227Q- and R227L-transfected cells. High-molecular-mass forms, likely to be dimers, were seen equally for wild-type and mutant receptors.
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The Western blot analysis that was consistent with the mutant CaSRs having achieved mature glycosylation (like wild-type) suggested they were appropriately trafficked to the plasma membrane. To approach this issue in an additional way and more directly analyze whether the CaSR mutants were expressed on the cell surface, fluorescence immunocytochemistry was performed on HEK293 cells transiently transfected with c-Myc-tagged wild-type and mutant CaSR cDNAs. The analysis was performed in 1) nonpermeabilized cells to detect cell surface staining only, indicating (if present) appropriate receptor maturation and trafficking to the plasma membrane, and 2) in permeabilized cells to assess the amount of receptor present intracellularly and undergoing maturation and trafficking to the plasma membrane.
Cells mock-transfected or transfected with untagged CaSR DNA showed no specific staining with the c-Myc antibody (data not shown). Strong staining was present at the cell surface of nonpermeabilized HEK293 cells transfected with c-Myc-tagged wild-type receptor (Fig. 4A). Permeabilization of such cells revealed further intracellular perinuclear staining associated with the endoplasmic reticulum and Golgi apparatus (Fig. 4B). Nonpermeabilized and permeabilized cells that had been transfected with either the R227Q or R227L mutants showed a similar pattern of staining, one to the other, and to that of the wild-type (Fig. 4, A and B).
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The ability of the mutant receptor to respond to extracellular calcium relative to the wild-type receptor was assessed using a trans-reporting system that measures the activity of Elk-1, an ETS domain transcription factor targeted by MAPK pathways. This experiment was repeated three times with identical results. A representative experiment is shown. The wild-type CaSR cDNA, when transiently expressed in HEK293 cells, showed a half-maximal response (EC50) of 3.7 ± 0.14 mM (mean ± SE; Fig. 5A). Both mutants showed significant rightward shifts in their dose-response curves relative to the wild type. However, the rightward shift was less marked for the R227Q mutant, with an EC50 of 7.9 ± 0.13 mM, relative to that for the R227L mutant, with an EC50 of 9.7 ± 0.12 mM (Fig. 5A). When equal amounts of wild-type and either R227Q or R227L mutant CaSR cDNAs were transiently coexpressed, in each case, the dose-response curves were rightward shifted to a position intermediate to that of the wild type alone and the mutant alone (Fig. 5B, Table 2, and data not shown).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Molecular analysis identified a heterozygous R227Q mutation in the proband and her affected relatives. This particular mutation has been described previously in an unrelated FBHH kindred (34). This family was one of those included in the original report of mapping of the FBHH locus to chromosome 3q (17). The mean serum calcium in the affected members was 11.8 mg/dl (2.95 mmol/liter) accompanied by normal levels of serum PTH and relative hypocalciuria. Therefore, the presentation was very similar to that of the family of the present report (see Table 2).
A heterozygous mutation at the same codon, but to a different amino acid, R227L, has also been reported (22). This was found in a girl with congenital hyperparathyroidism who presented soon after birth with respiratory distress, hypotonia, feeding difficulties, and bone deformities (35). Hypercalcemia and markedly raised serum PTH levels were present. Serum alkaline phosphatase was elevated, and the bony changes of primary hyperparathyroidism were evident on x-ray. At surgery, four hyperplastic parathyroid glands were removed, and the patient has been maintained on vitamin D metabolite therapy resulting in reversal of the bone abnormalities. Hence, the clinical presentation of the R227L mutation was markedly different from that of individuals harboring the R227Q mutation.
The CaSR is a member of group 3 of the G protein-coupled receptor superfamily in which agonists bind to a bi-lobed so-called Venus-flytrap (VFT) domain in the extracellular part of the receptor. The CaSR extracellular domain has several potential N-linked glycosylation sites and some of these must be glycosylated for receptor expression at the cell surface (36). Glycosylation is important for proper folding and trafficking of the protein. A model of the VFT domain of the CaSR based on the crystal structure of the related mGluR1 has been generated (37). The CaSR is a dimer, the monomers of which are linked by two intermolecular disulfide bonds within the extracellular domain. However, dimerization of the CaSR also involves noncovalent interactions (38, 39). Agonist binding to a cleft between two lobes leads to VFT closure and rotation about the dimer interface. Negatively charged amino acids are abundant in residues 215–251 within lobe 2 and could contribute to cationic ligand binding.
The functional importance of CaSR dimerization is shown by the complementarity of CaSR monomers, each with mutations in different receptor domains. For example, VFT mutants or intracellular COOH-terminal tail mutants have markedly reduced function as homodimers but when coexpressed as heterodimers have improved function (40). However, not all regions complement each other, for example, the cysteine-rich region linking the VFT to the seven-transmembrane domain (7TM) and the 7TM domain itself (41). Agonist-promoted VFT closure (active state) causes rotation of the monomers permitting the lobe 2 domains to move closer than in the open VFT (inactive state). It has been speculated that these ligand-activated changes cause movement of the cysteine-rich domain referred to above allowing it to communicate with the 7TM domain. Hence, amino acid 227 that is within lobe 2 at the dimer interface is in a very critical portion of the receptor with respect to ligand binding and the subsequent conformational changes that link to activation of the receptor. Several of the naturally occurring mutations, both inactivating and activating, are found within this region.
Functional studies have been reported for several of the naturally occurring CaSR mutants (28, 31, 42). CaSR cDNAs engineered to contain the mutations are transfected into human kidney cells. Immunoblot analysis of the cell extracts indicates whether the receptor is expressed as a protein and, if so, whether the amount, glycosylation, and dimerization status are normal. Ligand binding, receptor activation, and cell signaling properties are inferred from studying the changes brought about in the signaling pathways to which the CaSR couples in response to increases in extracellular calcium concentrations. Much useful information has been obtained in this way about how the CaSR normally works and how individual mutations can exert their deleterious effects in one of several different ways (28). With respect to the R227Q mutation of the present report, although the mutation itself has been described previously, no functional analysis had been undertaken. Therefore, in the present study, we have made a functional analysis of both the R227Q and R227L mutants.
Our analyses showed that both mutants were expressed in normal amount and underwent mature glycosylation and achieved cell-surface expression like the wild type. The functionality of CaSR mutants with respect to their ability to be activated by ligand and couple to intracellular signaling pathways has been carried out in different ways. Increases in intracellular calcium or inositol 1,4,5-trisphosphate production or, more recently, MAPK activity (30, 43) have been monitored in response to increases in extracellular calcium. In the present study, the R227L mutant demonstrated a rightward shift in MAPK responsiveness to extracellular calcium relative to wild type (Table 2). This can be compared with a previous functional analysis of the R227L mutant measuring increases in intracellular calcium transients in which similar EC50 values were obtained (42) (Table 2). Thus, the R227L mutant is markedly, but not absolutely, defective in ligand-activated cell signaling. The R227Q mutant has not been examined in this way before. In the present study, the R227Q also demonstrated a rightward shift in MAPK responsiveness to extracellular calcium increases relative to wild type. Therefore, it was impaired with respect to ligand-activated cell signaling but less so than the R227L mutant (Table 2).
For CaSR mutants, such as R227Q and R227L, that are heterozygously expressed and not impaired with respect to dimerization and trafficking to the cell surface, it would be predicted that dimer populations exist in the following ratios: wild-type homodimer, 25%; wild-type/mutant heterodimer, 50%; and mutant homodimer, 25%. In the present study, for both mutants, coexpression of equal amounts of wild-type and mutant CaSR produced a MAPK response intermediate to that of wild type or mutant alone. Hence, whereas the mutant/wild-type heterodimer is less effective than the wild-type homodimer, there is no evidence of synergy on the part of the mutant to cause the function of the heterodimer to more closely resemble that of the mutant homodimer.
With respect to the two different mutants examined here, how well does their functional analysis explain their markedly different clinical presentations? On the one hand, the R227L mutant, associated with the case of neonatal hyperparathyroidism, was less effective in signaling in response to the ligand relative to R227Q. More importantly, perhaps, in this case of a de novo mutation, the normal maternal calcium level would be read as a low calcium concentration by the defective calcium-sensing mechanism of the fetus resulting in a marked stimulus to fetal hyperparathyroidism. Hence, the disturbed maternal-fetal calcium homeostatic relationship is likely to have contributed significantly to the more severe presentation of the R227L mutation relative to the R227Q mutation we describe in a kindred with FBHH.
Other factors such as vitamin D status or general health of the mother may also play a role by modulating CaSR expression derived from the normal allele. In this regard, we have previously demonstrated that 1,25-dihydroxyvitamin D (44) and cytokines (45) are important regulators of CASR gene expression. In addition, there are several polymorphisms in the CASR gene, and some may be associated with serum calcium concentration (46). Therefore, it is possible that particular CASR polymorphisms influence the clinical presentation of some CASR mutations. This is an issue for future study.
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Acknowledgments |
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Footnotes |
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First Published Online November 30, 2004
Abbreviations: CaSR, Calcium-sensing receptor; FBHH, familial benign hypocalciuric hypercalcemia; 7TM, seven-transmembrane domain; VFT, Venus-flytrap.
Received September 9, 2004.
Accepted November 15, 2004.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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B pathway and B elements. J Biol Chem, in press
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15, not shown).