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Human Molecular Genetics Advance Access originally published online on June 1, 2006
Human Molecular Genetics 2006 15(14):2200-2209; doi:10.1093/hmg/ddl145
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly

Svetlana Pidasheva1,4, Michael Grant1,5, Lucie Canaff1,4, Oya Ercan7, Ujendra Kumar1,5,{dagger} and Geoffrey N. Hendy1,2,3,4,6,*

1 Department of Medicine, 2 Department of Physiology, 3 Department of Human Genetics, 4 Calcium Research Laboratory, 5 Fraser Laboratories and 6 Hormones and Cancer Research Unit, Royal Victoria Hospital, McGill University, Montreal, Canada QC H3A 1A1 and 7 Cerrahpasa Medical Faculty, Department of Paediatrics, Istanbul University, Istanbul, Turkey

* To whom correspondence should be addressed at: Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Room H4.67, Montreal, QC, Canada H3A 1A1. Tel: +1 5148431632; Fax: +1 5148431712; Email: geoffrey.hendy{at}mcgill.ca

Received April 6, 2006; Accepted May 30, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcium-sensing receptor (CASR) is a plasma membrane G protein-coupled receptor (GPCR) that plays a central role in regulating extracellular calcium ion concentrations. The CASR is expressed abundantly in the tissues and cells involved in calcium homeostasis, such as the parathyroid glands and in the cells lining the kidney tubule (1). This receptor is responsive to small changes in the circulating calcium concentration, and once activated, it inhibits parathyroid hormone (PTH) secretion and renal tubule calcium reabsorption.

The key role of this receptor as a ‘calciostat’ has been emphasized by the identification of naturally occurring inactivating or activating mutations in the CASR gene causing hypercalcemic or hypocalcemic disorders, respectively (2–4). Familial (benign) hypocalciuric hypercalcemia (FHH) is caused by heterozygous loss-of-function mutations, an asymptomatic condition in most cases (5,6). However, the homozygous form of such mutations causes neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism which occur within the first 6 months of life (7). So far, over 70 naturally occurring loss-of-function mutations have been reported (see the online database at http://www.casrdb.mcgill.ca) (8). Identification and analysis of these mutations has helped to better understand the structure and function of the receptor.

It has been known for some time that growth factor receptors of the tyrosine kinase type undergo ligand-induced dimerization at the cell surface. More recently, it has been appreciated that several GPCRs dimerize and it has been demonstrated that the CASR exists as a homodimer on the cell surface and that this is not ligand-induced (9). However, it is not clear where precisely dimerization of this receptor occurs.

In the present study, we describe the identification of novel mutations in the CASR in individuals with FHH or NSHPT. These two mutants (in addition to a previously described mutant) were analyzed with respect to cellular and plasma membrane expression, cell signaling, colocalization with endoplasmic reticulum (ER) and Golgi markers and dimerization status as assessed by photobleaching fluorescence resonance energy transfer (pbFRET). In this way, we showed that dimers of the CASR are already present in the ER and implicate dimerization as an important early event in the life history of this GPCR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Case 1
This family (Fig. 1) came to attention when the first child, a boy (VI-1), of related parents was admitted at 3 months of age with complaints of dyspnea, hypotonia, constipation, vomiting and failure to thrive. He was hypercalcemic (Table 1). This patient had hyperparathyroid bone disease and underwent total parathyroidectomy. He died from respiratory failure a month later, despite being normocalcemic. The second child (VI-2), a girl, was born a year later. She was admitted at 2.5 months of age and immediately underwent total parathyroidectomy and heterotopic autotransplantation. She had medical treatment for hypocalcemia and later hypercalcemia and became normocalcemic 4 months later. The parents (V-2 and V-6) are mildly hypercalcemic (Table 1) with low urinary calcium/creatinine clearance ratios (data not shown).


Figure 1451
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  		Figure 1. Pedigree of family 1 with FHH/NSHPT. Clinical status is indicated by open symbols (unaffected or status not known), hatched symbols (FHH) and solid symbols (NSHPT). Further clinical details are given in Table 1. Dotted symbols indicate neonatal death (with unknown serum calcium level). The proband is indicated by the arrow.

 


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  		Table 1. Biochemical characteristics of FHH/NSHPT family 1 members

 
Case 2
An individual who was diagnosed with FHH on the basis of modest hypercalcemia, inappropriately normal serum PTH levels and a low urinary calcium to creatinine clearance ratio.

Case 3
The proband of this family was diagnosed with NSHPT at birth (10). Before parathyroidectomy, his serum calcium level was 19 mg/dl and PTH concentration was >800 pg/ml (normal range 10–65). He is homozygous, and his parents heterozygous, for an R66C mutation in the CASR (11).

Identification of CASR mutations
Case 1
Direct sequence analysis of PCR-amplified CASR exons identified a homozygous mutation (R66H, CGT-CAT) encoded by exon 3 of the gene in the proband (VI-1) of the family. The sister (VI-2) was homozygous and both parents (V-2 and V-6) were heterozygous for the mutation that introduced an NcoI enzyme restriction site, not present in wild-type DNA, which was useful for mutation confirmation in the proband and analysis of family members. This change was not found in 100 CASR gene alleles from 50 unrelated normal individuals.

Case 2
Direct sequence analysis of PCR-amplified CASR exons in the FHH patient identified a heterozygous mutation in exon 7 such that an additional ‘T’ was inserted between codons 582 and 583, causing a frame shift and introduction of a stop codon at position 583 (N583X). This change was not found in 100 CASR gene alleles from 50 unrelated normal individuals.

Transfected R66H and N583X mutants demonstrate impaired cell signaling in response to extracellular increases in CASR agonists in HEK293 cells
The ability of the mutant receptors to respond to extracellular increases in CASR ligands relative to wild-type receptor was assessed in different systems. To test mitogen-activated protein kinase (MAPK) activity, two different assays were used. In a trans-reporting system that measures the activity of Elk-1, a transcription factor targeted by MAPK pathways, the wild-type CASR, when transiently expressed in HEK293 cells, exhibited a half-maximal response (EC50) of 3.9±0.15 mM (mean±SE) (Fig. 2A) to increasing calcium concentrations. In contrast, both the R66H and N583X mutants were unresponsive and the R66C mutant was poorly responsive even to very high calcium levels (Fig. 2A).


Figure 1452
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  		Figure 2. (A) Extracellular Ca2+-evoked increases in MAPK activity in HEK293 cells transiently transfected with either wild-type (WT) or mutant (R66C, R66H or N583X) CASR cDNA and a MAPK trans-reporting system. Values shown are the mean±SE of four estimations. (B) Extracellular Gd3+-evoked increases in intracellular Ca2+ in HEK293 cells (stably expressing apoaequorin) that were transiently transfected with either wild-type (WT) or mutant (R66C, R66H or N583X) CASR cDNA and the day after charged with coelenterazine-cp to generate holoaequorin. Luminescence values shown are the mean±SE of eight estimations.

 
Also, changes in the activation status of endogenous extracellular kinases 1 and 2 (ERK 1/2) in response to increasing extracellular calcium concentrations in HEK293 cells that had been transfected with the CASR cDNAs were assessed by a western blot assay of the phosphorylated and total ERK 1/2. This indicated that for the wild-type CASR, increasing the extracellular calcium concentration stimulated phosphorylation of ERK 1/2 with no change in total ERK 1/2 and tubulin. However, little or no activation of ERK 1/2 was found for the R66C, R66H or N583X mutants (data not shown).

In addition, increases in intracellular calcium transients in response to changes in the extracellular concentration of the CASR ligand, gadolinium (Gd3+), were monitored in HEK293 cells stably expressing apoaequorin that were then charged with coelenterazine to form holoaequorin. In cells transiently expressing the wild-type receptor, increases in luminescence occurred with increasing extracellular Gd3+ concentrations (EC50, 70±3.0 µM; mean±SE). However, the R66H and N583X mutants were completely unresponsive and the R66C mutant had much reduced responsiveness to Gd3+ (Fig. 2B).

Transfected R66H and N583X mutants demonstrate little cell-surface expression in HEK293 cells
To examine whether the mutant CASRs 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 and then permeabilized or not. Cells mock-transfected or transfected with empty vector or untagged CASR cDNA showed no specific staining with the c-Myc antibody (data not shown). Strong cell-surface staining was present in non-permeabilized HEK293 cells transfected with the epitope-tagged wild-type receptor (Fig. 3A). Permeabilization of such cells revealed further intracellular perinuclear staining associated with the ER and Golgi apparatus (Fig. 3B). In contrast, non-permeabilized cells that had been transfected with mutant CASR cDNAs demonstrated either no (R66H, N583X) or very little (R66C) cell-surface staining, respectively (Fig. 3A). However, permeabilized cells transfected with these mutants demonstrated intracellular perinuclear staining similar in amount to cells transfected with the wild-type (Fig. 3B). The HEK293 cell transfection and fluorescence immunocytochemistry analysis showed that although all the CASR mutants were deficient with respect to cell surface expression, they were present intracellularly.


Figure 1453
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  		Figure 3. Plasma membrane and intracellular expression of wild-type and mutant CASRs in transfected HEK293 cells. Fluorescence immunocytochemistry and confocal microscopy were performed on HEK293 cells transiently transfected with wild-type (WT) or mutant (R66H, R66C, N583X) c-Myc-tagged CASR cDNA. Immunostaining was done with c-Myc monoclonal antibody 9E10, and detection was made with goat anti-mouse FITC-conjugated secondary antibody. Examples of fields of non-permeabilized (A) and permeabilized (B) cells are shown.

 
CASR mutants are found in the ER but not in the Golgi apparatus
To identify where the CASR mutants are present intracellularly, colocalization studies were carried out with specific markers for the ER (anti-calnexin antibody) or the Golgi apparatus (wheat germ agglutinin conjugated to fluorescein) and the CASR (anti-c-Myc antibody conjugated to Cy3) in HEK293 cells transiently transfected with either wild-type CASR or one of the mutant CASR cDNAs. In the cells expressing the wild-type CASR, localization of the CASR to both organelles was demonstrated (Fig. 4A); however, the CASR mutant, R66H, although being present in the ER was not found in the Golgi apparatus (Fig. 4B). A similar observation was made for the other CASR mutant, R66C (data not shown).


Figure 1454
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  		Figure 4. CASR and ER and Golgi apparatus colocalization. Fluorescence immunocytochemistry and confocal microscopy were performed on HEK293 cells transiently transfected with (A) wild-type (WT) or (B) R66H mutant c-Myc-tagged CASR cDNA. Representative fields are shown for cells incubated with (left panels) anti-calnexin antibody followed by FITC-labeled IgG for ER staining (green) or wheat germ agglutinin conjugated to fluorescein for Golgi apparatus staining (green). Middle panels: c-Myc-Cy3-conjugated monoclonal antibody was added to visualize the CASR (red). Merging of the right and middle panels is shown in the right panels (yellow indicates colocalization).

 
Transfected R66H and R66C mutants demonstrate no or little of the mature (160 kDa) glycosylated form although dimerization is unimpaired
To examine whether the mutant CASRs are expressed normally and exhibit similar molecular species as wild-type CASR, western blot analysis of extracts of HEK293 cells transiently transfected with either the c-Myc-tagged wild-type (positive control) or mutant CASR cDNAs or empty vector (negative control) was carried out. The CASR molecular species were revealed on immunoblots with an antibody against the c-Myc epitope tag. Under the particular western blotting conditions used, the wild-type CASR exists in both monomeric and dimeric forms: the monomeric non-glycosylated species is 120 kDa (but is not present in sufficient amount to be revealed on the immunoblot), the core glycosylated (immature) species is 140 kDa and the mature, fully glycosylated species, is 160 kDa (Fig. 5A). Immature glycosylation occurs early in the ER, whereas mature glycosylation occurs only later after trafficking to the Golgi apparatus. Dimeric (or oligomeric) forms of the receptor migrate at a size of >280 kDa on the immunoblot (Fig. 5A). Both monomeric and dimeric species were present in the R66H and R66C mutant-transfected cells; however, virtually none or little of the 160 kDa species was evident for the R66H or R66C mutants, respectively (Fig. 5A). For the N583X mutant, a single monomeric species of ~75 kDa was observed with no dimers present (Fig. 5A). The ~75 kDa species was reduced to ~65 kDa by endoglycosidase H (Endo H) treatment revealing it to represent an immature core-glycosylated form (data not shown). Under these conditions, no mature glycosylated species was detected indicating that, if occurring, appropriate trafficking of any N583X mutant protein to the cis-Golgi and release into the medium was only taking place at an extremely low level. Consistent with this hypothesis, although western blot analysis of conditioned medium of N583X-transfected cells did identify a single ~85 kDa Endo H-resistant species (therefore representing a mature glycosylated form), it was present in an amount some 200-fold less than a control of exogenously expressed progranulin representing a known constitutively secreted glycoprotein of similar size to the CASR N583X mutant (data not shown). Hence, the N583X protein appearing in the medium likely represented a very minor fraction of the total that had escaped the quality control mechanisms that retained the vast majority of the N583X mutant within the cell. By biotin cell surface labeling of HEK293 cells stably expressing the wild-type CASR, followed by immunoprecipitation and western blotting, CASR dimers (oligomers) were identified as the molecular species present on the cell surface (Fig. 5B).


Figure 1455
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  		Figure 5. (A) Expression levels of wild-type and mutant CASRs in HEK293 cells. Western blot analysis was performed on extracts of HEK293 cells either empty-vector transfected (vector) or transiently transfected with wild-type (WT) or mutant (R66H, R66C, N583X) c-Myc-tagged CASR cDNA. CASR proteins were stained with c-Myc monoclonal antibody 9E10 and endogenous proteins (for loading control) were stained with ß-tubulin antibody. (B) HEK293 cells stably expressing c-Myc-tagged wild-type CASR were subjected to cell-surface biotin labeling followed by immunoprecipitation and western blotting with the c-Myc monoclonal antibody and ß-tubulin antibody.

 
pbFRET microscopy
To further characterize the dimerization status of the wild-type and mutant CASRs, pbFRET microscopy was used. The FRET technique is a spectroscopic method for monitoring the energy transfer from an excited molecule (the donor) to another molecule (the acceptor) and it occurs when the absorption spectrum of the acceptor overlaps with the emission spectrum of the donor. When used for measuring GPCR protein–protein interactions, the technique can discriminate between receptors localized intracellularly as opposed to those that are cell-surface associated. The competitive nature of FRET and photobleaching (irreversible loss of a fluorophore's capacity to fluoresce) allows indirect measurement of FRET via its effect on donor photobleaching. In HEK293 cells transfected with the c-Myc-tagged CASR R66H mutant, the staining patterns obtained with anti-c-Myc antibodies conjugated to either donor (FITC) or acceptor (Cy3) fluorophores colocalized in intracellular compartments (Fig. 6A). The photobleaching decay and the associated time constants of the donor molecule were calculated from experiments in the absence and presence of the acceptor molecule, respectively (Fig. 6B and C). Any retardation in photobleaching on exposure to light indicates that the acceptor molecules are in sufficient proximity to donor molecules to act as acceptors for energy transfer. Given that the two fluorophores are associated with different receptor molecules, receptor association is indicated. A decrease in photobleaching decay rate was observed in the presence of the acceptor molecule (Fig. 6C), showing that the donor and acceptor molecules were within 100 Å of each other, indicative of protein–protein interaction. Similar experiments were conducted with HEK293 cells transfected with either wild-type CASR or the R66C and N583X mutants. The time constants for each condition were averaged over many cells and converted into FRET efficiencies (Fig. 7). Both cell-surface and intracellular wild-type CASR and intracellular CASR R66H and R66C mutants exhibited relatively high FRET efficiencies (~17–20%), an indication that these receptors all exist as dimers. The exception was the CASR truncation mutant N583X, with a relatively low FRET efficiency (~3%), inconsistent with this receptor molecule existing in a dimeric form.


Figure 1456
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  		Figure 6. Confocal microscope images and representative histogram time constant plots from pbFRET microscopy of HEK293 cells transiently expressing the c-Myc-tagged CASR R66H mutant. (A) Cells were incubated with primary anti-c-Myc mouse monoclonal antibody followed by (i) secondary anti-mouse fluorescein-conjugated antibody shown in green or (ii) anti-mouse Cy3-conjugated antibody shown in red and (iii) colocalization of the two images shown in yellow. (B) Photobleaching of fluorescein (donor) in the absence of Cy3 (acceptor). Top: cells were treated with the appropriate antibodies, fixed and followed by donor photobleaching with 488 nm light. A sequence of 20 timed images was captured (one image every 4 s with exposure time of 3 s), and a subset is shown. Bottom: the time constants displayed in the histogram bar plot were calculated on the basis of a pixel-by-pixel analysis of a selected intracellular area. The mean time constant of 16.8 s is shown as a black bar calculated by averaging the time constants from a Gaussian distribution. (C) As in (B), but with photobleaching of fluorescein (donor) in the presence of Cy3 (acceptor). Top: there is an obvious visual delay in photobleaching of the donor in the presence of the acceptor. Bottom: the histogram time constant bar plot displays the increased mean time constant (21.6 s).

 


Figure 1457
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  		Figure 7. Effective FRET efficiencies were determined as described in the legend to Fig. 6 and in Materials and Methods in HEK293 cells transiently transfected with wild-type CASR (wt) or CASR mutants (R66H, R66C, N583X) and permeabilized (P) or not (NP).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The fact of dimerization of GPCRs has only been appreciated relatively recently, and the mechanisms underlying the process of dimerization and its underlying functional significance are still being worked out for the different subgroups within the GPCR superfamily. GPCR dimerization could potentially affect trafficking to the plasma membrane, ligand binding, activation, signaling and desensitization and internalization. Some GPCRs dimerize only on the cell surface upon ligand activation, whereas others dimerize intracellularly prior to reaching the cell surface. Agonist-induced oligomerization has been shown for receptors such as those for somatostatin (12,13), thyrotropin-releasing hormone and gonadotropin-releasing hormone (14). For the CASR, evidence has been presented that it is present as a dimer on the cell surface, but that the calcium ligand does not affect the dimerization status (9).

In the present study, we identified two novel inactivating CASR mutations R66H and N583X in FHH and/or NSHPT patients. CASR mutants harboring these mutations along with another one representing the previously identified R66C mutation were characterized functionally and compared with the wild-type CASR in a number of ways. The CASR can couple to different types of G protein and activate multiple signaling pathways. Coupling to the pertussis, toxin-insensitive Gq/11 stimulates phospholipase C-mediated inositol trisphosphate (IP3) formation with intracellular Ca2+ mobilization and PKC activation, which contributes to stimulation of the MAPK cascade (15). Some evidence has been presented that activation of the MAPK pathway is relevant to the control of parathyroid secretory function by the CASR (16,17). In the present study, changes in intracellular Ca2+ (using an aequorin assay) and MAPK activation (both exogenous and endogenous) were measured in response to increases in the CASR agonists Gd3+ or Ca2+. All three CASR mutants were defective with respect to cell signaling when compared with the wild-type. The complete lack of activity would be expected in the case of the N583X truncation mutant. The absent or markedly reduced activity of the R66H and R66C mutants, respectively, is consistent with the fluorescence immunocytochemical analysis of non-permeabilized cells that showed no or little cell-surface staining for all the mutants compared with the wild-type CASR.

However, the immunocytochemistry confocal microscopy of permeabilized cells did show that all three mutants were well expressed intracellularly similar to the wild-type. Therefore, these particular mutants belong to a class of CASR mutants that are retained within the cell and are unable to traffic normally to the cell surface. Previously, we had shown that other inactivating mutations, G549R and C850_851ins/fs, were only present intracellularly and not at the cell surface (18).

The CASR undergoes core (immature) N-linked glycosylation in the ER. Once properly folded, the core-glycosylated receptor transits to the Golgi apparatus where it undergoes mature glycosylation and is then expressed at the cell surface. The N-glycosylation with complex carbohydrates is important for cell-surface expression of the CASR but is not critical for signal transduction (19). Enzymatic deglycosylation experiments on extracts of HEK293 cells transfected with wild-type or mutant CASRs have defined the molecular species observed by immunoblotting analysis (18,20). The 160 kDa species is the fully glycosylated (mature) form, whereas the 140 kDa species is the immature core-glycosylated form. From the immunoblot analyses of the present studies, it is clear that the N583X truncation mutant does not achieve mature glycosylation essentially at all and that the R66H and R66C mutants undergo either no or little mature glycosylation, respectively. This is consistent with the results of the fluorescence immunocytochemistry and indicate that the mutants were all defective in trafficking to the plasma membrane.

Colocalization experiments were performed with the wild-type CASR and the mutants with markers specific for the ER and Golgi apparatus. Although the wild-type CASR was clearly localized to both organelles, none of the mutants was found in the Golgi, but only in the ER, indicating an impairment in their cellular trafficking.

Many studies have documented, by immunoblot analysis of parathyroid or kidney tissues or HEK293 cells transfected with the wild-type CASR cDNA, molecular species of >280 kDa. These represent dimers (or higher order oligomers) that are resistant to complete denaturation under the conventional SDS–PAGE conditions used. The CASR dimerizes through multiple types of intermolecular interactions including disulfide linkages involving C129 and C131 (21,22) and non-covalent interactions between L112 and L156 on each protomer, all within the extracellular domains (23). Mutation of some but not all of these interacting sites does not disrupt dimer formation but may lead in some cases to enhanced cell-signaling activity (23). In the present study, by immunoblot analysis of extracts of HEK293 cells transfected with the N583X truncation mutant cDNA, no dimeric form of the receptor was evident, only a monomeric species of ~75 kDa. This would be consistent with the view that, in addition to the interactions between the dimer interface in the extracellular domains (~600 amino acids) of the two protomers, non-covalent interactions involving other parts of the molecule are likely to be important (24,25). In contrast, the R66H and R66C mutants, although having almost no mature monomeric forms, had significant amounts of SDS-resistant dimeric forms similar to wild-type. By biotin cell-surface labeling of HEK293 cells that stably express the c-Myc-tagged CASR followed by immunoprecipitation with c-Myc antibody and immunoblot with streptavidin, it was confirmed that the CASR is present predominantly at the cell surface as a dimer.

With the naturally occurring CASR mutants that are retained intracellularly, two of which (R66H and R66C) that could dimerize and one (N583X) that could not, the issue of intracellular dimerization of the CASR was explored further by using the biophysical pbFRET method. In this context, the N583X mutant is valuable as a negative control. The FRET efficiency (E) depends on the distance between and the orientation of the chromophores, with enhanced resolution up to 100 Å. Hence, E is related to the level of dimerization. A two-state model is assumed in which receptors exist in one of two populations in either a monomeric or a dimeric state. FRET occurs only in a receptor dimer composed of one donor and one acceptor-labeled receptor. When donor and acceptor molecules are close enough, the acceptor fluorophore offers an alternate route for the deactivation of the energetically excited state of the fluorophore, which leads to slower photobleaching, an intrinsic property of a fluorophore characterized by the fading of the fluorescent signal when continuously exposed to excitation light of the donor molecule (13).

In the present study, a c-Myc epitope-tagged CASR was used. Only one monoclonal anti-Myc monoclonal antibody molecule can be bound per receptor molecule. If when receptors are incubated with both FITC- and Cy3-conjugated monoclonal antibodies, donor and acceptor, respectively, and a slower rate of photobleaching is observed in comparison to when only the donor FITC-conjugated antibody is used, then dimerization is indicated. The present experiments demonstrated that R66H and R66C mutants present in the ER, exist, like wild-type CASR, as dimers.

Dimers of the CASR have been identified in detergent extracts of rat kidney medulla (26), and the use of the bioluminescence resonance energy transfer technique in cells expressing GFP-tagged CASR has indicated the presence of dimers (27). In the present study, we provide firm evidence of constitutive dimerization of the CASR in the ER. In addition, the finding of dimerization of mutant CASRs that are retained in this organelle suggests that although dimerization of the CASR may be necessary, it is not sufficient for the exit of the receptor from the ER and trafficking to the cell surface.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
All subjects gave informed consent for the study that was approved by the respective institutional Ethics Committees.

Reagents
The c-Myc 9E10 mouse monoclonal antibody was from Santa Cruz Biotechnologies, the calnexin antibody (28) was kindly provided by Dr Eric Chevet (McGill University), the wheat germ agglutinin conjugated to fluorescein was as described (29), PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) antibody kits were from Cell Signaling Technology, Beverly, MA, USA, the HA-tagged progranulin expression vector was kindly provided by David Baranowski and Dr Hugh P.J. Bennett (McGill University) and the pGL3 promoter plasmid was from Promega.

Sequence analysis of the CASR gene
Leukocyte DNA was isolated using standard methods. Exons 2–7 of the CASR gene were amplified as described (18). Gel purified PCR products were directly sequenced.

Site-directed mutagenesis
The Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) 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, USA) were cultured in 100 mM plates and transfected with human CASR cDNA (8 µg) using PolyFect transfection reagent. Forty-eight hours after transfection, cells were harvested for total cellular protein extraction and western blot analysis of total cell extracts was performed with the c-Myc 9E10 monoclonal antibody (18). All experiments were repeated at least three times and membranes were stripped and re-probed with ß-tubulin mouse monoclonal antibody as a loading control.

Stable HEK293 cell lines expressing cDNA for wild-type CASR, empty vector and/or apoaequorin
HEK293 cells (that do not express CASR) were cultured in DMEM growth medium containing 10% fetal bovine serum. Cells were transfected with plasmid pcAEQ (30) that has the apoaequorin gene inserted in pcDNA3.1 (kindly provided by Drs A.D. Conigrave and A.H. Franks, University of Sydney, Australia), wild-type CASR or empty vector pcDNA3.1. Seventy-two hours after transfection, a medium containing 500 µg/ml of G418 was used to select only plasmid expressing cells. Non-transfected cells were used as a control. The selection medium was changed every 3 days and resistant colonies were maintained for 2 weeks. Several G418-resistant well-isolated colonies were selected for each cell line. The expression of apoaequorin, CASR and GAPDH was assessed by RT–PCR, in the case of the CASR, using primers located in different exons (4 and 6), and western blot analysis (data not shown). Mock-transfected or empty vector-transfected HEK293 cells were used as a control.

Cytoplasmic-free Ca2+ changes measured by aequorin luminescence assay
HEK-293 cells stably expressing aequorin were transiently transfected with wild-type or mutant CASR cDNA and pGL3 promoter plasmid and aliquoted into 96-well plates 24 h later. The next day, cells were rinsed once and charged for 1 h at 37°C in a buffer [20 mM HEPES, pH 7.4, 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 0.1% bovine serum albumin (BSA), 0.1% dextrose] containing 5 µM coelenterazine (cp form, Molecular Probes, Inc., Eugene, OR, USA) to form holoaequorin (30). The charging solution was then replaced with solution containing a low calcium concentration (20 mM HEPES, pH 7.4, 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% dextrose, 0.1% BSA). Transfected cells were exposed to increasing Gd3+ concentrations (25–400 µM) and luminescence measurements were made (Fluostar Optima; BMG Labtech). Light emission was recorded at 0.5 s intervals for 30 s. The maximum response (for either wild-type or mutant CASR cDNA) was normalized to that with the empty vector pcDNA3.1. All the experiments were done with eight replicates per sample and repeated three times. Normalization for transfection efficiency was made to luciferase activity (pGL3) assay.

MAPK assays
MAPK activity induced by activation of the CASR was assessed in two ways. Amounts of endogenous phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK 1/2) were assessed as described (31). In brief, HEK293 cells were transiently transfected with wild-type or mutant CASR cDNAs in six well plates. Forty-eight hours later, cells were serum-starved for 18 h and the medium was renewed for 2 h before cells were treated with various CaCl2 concentrations (0.5–10 mM) for 5 min. Cells were lysed with a buffer containing sodium ortho-vanadate and complete protease inhibitor tablets. Whole cell extract proteins were separated by 10% SDS–PAGE and phosphorylated and total ERK 1/2 were detected using the PhosphoPlus p44/42 MAP Kinase (Thr202/Tyr204) antibody Kit (Cell Signaling Technology) according to manufacturer's instructions.

Exogenous MAPK activity was assessed as described (32). 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) 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 phosphate-buffered saline (PBS) and lysed on ice. Luciferase activity was measured using 45 µl cell lysate and D-luciferin using a Fluostar Optima. Luciferase activity was normalized to ß-galactosidase activity.

Fluorescence immunocytochemistry and confocal microscopy
HEK293 cells were transiently transfected with either c-Myc-tagged wild-type or mutant CASR cDNA as described (33). 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 5% goat serum for 1 h and then at 4 C overnight with 9E10 c-Myc monoclonal antibody at a 1:250 dilution. Washed cells were incubated for 1 h with a goat anti-mouse FITC-conjugated antibody (Molecular Probes, Inc.). Slides were mounted with mount medium and dried overnight at room temperature. Confocal images of labeled cells were acquired with a Zeiss LSM 510 META laser-scanning microscope (Carl Zeiss, Jena, Germany) using a 60x oil immersion lens. FITC fluorescence was visualized using a singletrack mode with laser excitation (488 nm) and emission (LP 505) filter sets.

For the ER and Golgi colocalization studies, the protocol was similar to that described above except, after permeabilization, cells were washed and incubated in 10% goat serum for 1 h and then incubated either with affinity-purified anti-calnexin antibody ({alpha}C4) (28), followed by FITC-labeled donkey anti-rabbit IgG for ER staining, or with wheat germ agglutinin conjugated to fluorescein for Golgi apparatus staining (29). In both cases, c-Myc-Cy3-conjugated monoclonal antibody was added to visualize the CASR. For ER staining, cells were washed and then incubated with secondary antibody for 1 h and mounted and visualized by confocal microscopy.

Biotinylation of cell surface CASR and immunoprecipitation
HEK293 cells stably transfected with either the c-Myc-tagged wild-type CASR construct or the empty vector were treated as described (34). Cell-surface proteins of the intact cells were labeled with membrane-impermeant Biotin-7-NHS using a Cellular Labeling Kit (Roche Molecular Biochemicals). Cells were washed once with PBS and treated with 50 µg/ml Biotin-7-NHS in biotinylation buffer for 15 min at room temperature. Reactions were stopped by adding 50 mM NH4Cl and incubating on ice. The cells were washed twice with PBS and solubilized with 1 ml of lysis buffer per well containing 50 mM iodoacetamide and protease inhibitor mixture.

For immunoprecipitation, total protein of the whole cell lysate was incubated with a protein A-agarose suspension for 3 h at 4°C. Beads were pelleted by gravity centrifugation and supernatants were transferred to fresh tubes. After adding c-Myc 9E10 monoclonal antibody, the mixtures were incubated for 1 hour at 4°C. Protein A-agarose was added and incubation continued overnight at 4°C. Complexes were centrifuged and supernatants removed. The beads were washed three times and gel-loading buffer was added and proteins denatured at 100°C for 3 min. After centrifugation, aliquots of the supernatants were analyzed by electrophoresis through SDS 4–12% gradient gels. Gels were blotted onto PVDF membranes, incubated with blocking buffer to prevent non-specific binding of conjugate or substrate. The biotin-labeled proteins were reacted with a streptavidin-POD enzyme conjugate and visualized with a western blotting chemiluminescent reagent.

Immunocytochemistry and pbFRET microscopy
For pbFRET microscopy, HEK293 cells were transiently transfected with either c-Myc-tagged wild-type or mutant CASR cDNAs. Forty-eight hours later, cells were fixed with 4% paraformaldehyde in PBS on ice for 20 min. Cells were permeabilized with 0.2% Triton X-100 for intracellular localization of the receptors. Immunocytochemistry was performed with monoclonal anti-c-Myc antibody conjugated to either FITC or Cy3 corresponding to donor or acceptor, respectively, before being subjected to pbFRET analysis (12,35). The effective FRET efficiencies (E) were calculated according to the average photobleaching time constant of the donor obtained in the absence ({tau}ave D–A) and presence of the acceptor ({tau}ave D+A) using the equation E=1–({tau}D–A/{tau}D+A)x100 (12).


   ACKNOWLEDGEMENTS
 
We thank all family members for their participation, Drs S. Hatemi, E. Adal, D. Yardimci, E. Savgan, R. Kodakoglu, M. Karperien and S.E. Papapoulos for providing patient samples and/or clinical details, Dr E.M. Brown for help with the clinical evaluation of Case 1, Bing Yang and Irina Mosesova for technical support, Dr Stephane A. Laporte and Delphine Fessart for facilitating the confocal microscopy studies, Drs A.D. Conigrave and A.H. Franks for providing the apoaequorin plasmid and Drs Eric Chevet and Eric A. Shoubridge for critical review of the manuscript. This work was supported by the Canadian Institutes of Health Research (CIHR) Grants MOP-57730 (to G.N.H.) and MOP-74465 (to U.K.). S.P. was a recipient of a studentship from the McGill University Hospital Centre Research Institute and a scholarship from the CIHR Strategic Training Program in Skeletal Health. L.C. was the recipient of a postdoctoral fellowship from the Kidney Foundation of Canada.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Faculty of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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