Calcium Signaling Regulates Trafficking of Familial Hypocalciuric ...

ORIGINAL

RESEARCH

Calcium Signaling Regulates Trafficking of Familial Hypocalciuric Hypercalcemia (FHH) Mutants of the Calcium Sensing Receptor Michael P. Grant, Ann Stepanchick, and Gerda E. Breitwieser Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2604

Calcium-sensing receptors (CaSRs) regulate systemic Ca2⫹ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2⫹ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2⫹, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2⫹ signaling, which were substantially rescued by coexpression with wildtype CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2⫹ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSR⌬868 robustly rescued trafficking and Ca2⫹ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2⫹ response when extracellular Ca2⫹ is elevated and argues that Ca2⫹ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane. (Molecular Endocrinology 26: 2081–2091, 2012)

TH regulates organismal Ca2⫹ homeostasis by modulating Ca2⫹ dynamics in bone and kidney (1). PTH synthesis and secretion by the parathyroid gland are controlled by the calcium-sensing receptor (CaSR), a family C/3 G protein-coupled receptor (GPCR), which is activated by small changes in serum Ca2⫹ (2). The critical role for CaSR in regulation of PTH secretion and organismal Ca2⫹ homeostasis was suggested by knockout mouse models (3, 4) and confirmed by identification of mutations that reduce CaSR signaling in patients (5, 6). Mouse models suggest both alleles contribute to normal CaSR function (3, 4). In humans, loss-of-function mutations in

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a single CaSR allele cause benign familial hypocalciuric hypercalcemia (FHH) (7), whereas mutations in both alleles (or distinct mutations in two alleles) result in neonatal severe hyperparathyroidism (NSHPT) (5). CaSR is assembled in the endoplasmic reticulum (ER) as a disulfide-linked dimer (8). The large (⬃600 residues) extracellular domain (ECD) contains agonist (Ca2⫹, diand trivalent cations) and allosteric modulator (aromatic amino acids, glutathione) sites, and the heptahelical domain has binding pocket(s) for allosteric drugs, both agonists (calcimimetics) and antagonists (calcilytics) (9). The more than 200 loss-of-function mutations that have been

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/me.2012-1232 Received July 12, 2012. Accepted September 21, 2012. First Published Online October 17, 2012

Abbreviations: ADIS, Agonist-driven insertional signaling; BS-wt, chimera of wt CaSR; CaSR, calcium-sensing receptor; CFP, cyan fluorescent protein; ECD, extracellular domain; ER, endoplasmic reticulum; FHH, familial hypocalciuric hypercalcemia; FRET, fluorescence resonance energy transfer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HA, hemagglutinin; HEK, human embryonic kidney; nFRET, net FRET; NSHPT, neonatal severe hyperparathyroidism; TIRFM, total internal reflection microscopy; wt, wild type; YFP, yellow fluorescent protein.

Mol Endocrinol, December 2012, 26(12):2081–2091

mend.endojournals.org

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Calcium Signaling Rescues FHH Mutants of CaSR


metics can promote proper folding and/or enhanced plasma membrane targeting of FHH mutants, significantly increasing signaling (13–18). CaSR signals via multiple pathways, but Gq-mediated activation of phospholipase C␤ leading to increases in intracellular Ca2⫹ is a nearly ubiquitous response (1, 2, 19). Extracellular Ca2⫹-mediated activation of CaSR is highly cooperative, with small changes in extracellular Ca2⫹ (0.2– 0.5 mM) leading to Ca2⫹ oscillations, whereas larger changes in extracellular Ca2⫹ induce steady state increases in intracellular Ca2⫹ (20 –22). We have recently identified a unique mechanism that intimately links CaSR signaling and trafficking, i.e. agonist-driven insertional signaling (ADIS) (23). Elevated extracellular Ca2⫹ initiates a signaling-dependent increase in anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2⫹ is elevated. Endocytosis, in contrast, is constitutive and not modulated by extracellular Ca2⫹. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. Our experimental approach allows us to simultaneously monitor dynamic changes in plasma membrane levels of CaSR and net changes in intracellular Ca2⫹. Furthermore, the use of a chimeric CaSR construct allows us to explicitly define changes in plasma membrane levels of mutant or wild-type CaSR in the presence of nonchimeric partners. Here we compare the trafficking and Ca2⫹ signaling defects of selected FHH/NSHPT mutants and demonstrate that signalFIG. 1. ADIS of CaSR. A, Model for the construct used in these studies, having a minimal ing by wild-type CaSR rescues CaSR bungarotoxin binding site (BgTx) followed by Super Ecliptic pHluorin (SEP) inserted after the mutant trafficking. signaling peptide (23). B, HEK293 cells expressing BS-wt were imaged by TIRFM (SEP identified in patients with FHH/NSHPT are largely clustered within the extracellular and heptahelical domains, although a few mutations and deletions have been identified in the large carboxyl terminus (10). Reduced function of CaSR mutants can be attributed to folding defects, which increase targeting to the proteasome as part of endoplasmic reticulum quality control, more subtle folding defects, which limit exit of mutants from the endoplasmic reticulum, and/or mutations, which permit plasma membrane targeting but reduce the efficacy of Ca2⫹/agonists to activate signaling (11, 12). Discrimination of the mechanistic basis for the defect(s) causing reduced CaSR signaling may allow functional rescue of mutants. Indeed, recent studies have shown that calcimi-

fluorescence) as described in Materials and Methods. Representative images (488 nm laser illumination) are shown in 0.5 and 10 mM Ca2⫹ bath solutions and after return to 0.5 mM Ca2⫹ bath. C, Cells transfected with BS-wt and loaded with Fura Red were imaged by TIRFM and wide-field microscopy. Extracellular Ca2⫹ was increased from 0.5 to 10 mM as indicated. Images, taken at 15-sec intervals were analyzed with ImageJ, and values for both plasma membrane SEP fluorescence (top, green symbols) and Fura Red fluorescence (bottom, red symbols) were plotted as mean ⫾ SE (n ⫽4 cells). D, Experiments as in C, monitoring SEP fluorescence, with step changes in extracellular Ca2⫹ as indicated. Extracellular Ca2⫹ concentrations were 1, 2.5, 5, 10, 20, and 30 mM, indicated in color range from black through light blue. Images taken at 10-sec intervals; normalized intensities plotted as mean ⫾ 2⫹ SE, with n ⫽4 cells at each Ca concentration. E, Calculated steady-state responses at each extracellular Ca2⫹ concentration in D were fitted to a sigmoidal dose-response relation to determine the EC50 of 3.4 ⫾ 0.3 mM (maximum 149 ⫾ 2%).

Materials and Methods DNA constructs Human CaSR with amino-terminal additions of the FLAG epitope, a minimal bungarotoxin binding site and Super Ecliptic pHluorin (BS-wt) (23), served as the background for generation of FHH mutants (R227Q, E297K, C395R, R795W,


P798T, A804D), using primers described for generation of the mutants in the FLAG-CaSR background (14). FLAG-tagged versions of wild-type (wt), CaSR⌬868, and G143E were generated as described (14, 24). Hemagglutinin (HA)-CaSR (HA-wt) was generated by replacing the FLAG epitope with the HA epitope by PCR. FLAG-CaSR-cyan fluorescent protein (CFP) (CaSR-CFP) was generated by removing the stop codon and fusing CFP at the carboxyl terminus of FLAG-CaSR in pcDNA3.1. All constructs were verified by sequencing (Genewiz, South Plainfield, NJ).

Cell culture and transfection Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were maintained in MEM


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supplemented with 10% fetal bovine serum and penicillin/streptomycin in 5% CO2. Cells were transfected with FuGENE HD (Roche Applied Science, Mannheim, Germany) using the manufacturer’s protocol.

Immunoprecipitation and Western blotting HEK293 cells seeded in six-well plates and transiently transfected with CaSR constructs were cultured for 72 h and processed for immunoprecipitation with monoclonal anti-green fluorescent protein (GFP) (Abcam, Cambridge, MA; ab1218) or monoclonal anti-HA (Abcam; ab16918) antibodies plus protein-G-agarose (Invitrogen, Carlsbad, CA; 15920-010). Samples (sodium dodecyl sulfate loading buffer plus dithiothreitol, 30 min at room temperature) were electrophoresed on 4 –15% gradient Tris-HCl gels (Bio-Rad Laboratories, Hercules, CA) and transferred to polyvinyl difluoride membranes. Immunoblots of lysates or immunoprecipitates were probed with polyclonal anti-HA (SigmaAldrich, St. Louis, MO; H6908), polyclonal anti-GFP (Abcam; ab290), polyclonal anti-CaSR [LRG epitope (25) custom generated by Genemed Synthesis, Inc., South San Francisco, CA] or polyclonal antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma; G9545) antibodies and secondary antibody (enhanced chemiluminescence, horseradish peroxidase conjugated, antirabbit, GE Healthcare, Indianapolis, IN) as indicated in the figure legends. SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL; 34080) was used to visualize proteins on a Fuji Film LAS-4000 Imager (Tokyo, Japan).

Total internal reflection microscopy (TIRFM)

FIG. 2. ADIS of FHH mutants of CaSR. A, Model of the CaSR dimer showing locations of mutants. HEK293 cells transiently expressing BS-CaSR-mutants, R227Q (B), E297K (C), C395R (D), R795W (E), P798T (F), or A804D (G) and loaded with Fura Red were perfused with 30 mM Ca2⫹ bath solution (from baseline of 0.5 mM) for the indicated interval. Images were analyzed with ImageJ and plotted as mean ⫾ SE (n ⫽4 cells).

HEK293 cells grown on 22-mm glass coverslips coated with human fibronectin (Millipore, Billerica, MA) were transfected with the indicated constructs. After 72 h, media were replaced with osmolality matched 0.5 mM Ca2⫹ bath solution plus 0.1% BSA and incubated for 30 min. TIRFM images were collected on a Nikon TE2000-E microscope (Tokyo, Japan) equipped with a ⫻60, 1.45 NA objective, Perfect Focus, and a TIRF-2 illuminator, using laser lines at 488 and 594 nm. Both image acquisition and adjustment of TIRFM angles for each wavelength were automated with ␮Manager (National Institutes of Health, Bethesda, MD). Images were captured with a Photometrics CoolSNAP HQ2 charge-coupled device camera (Tucson, AZ) using appropriate emission filters mounted on a motorized shutter wheel (Ludl, Hawthorne, NY). For intracellular Ca2⫹ imaging, cells were incubated

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TABLE 1.



Ca2⫹-ADIS relations of CaSR FHH mutants

Receptor BS-wt BS-R227Q BS-E297K BS-C395R BS-R795W BS-P798T BS-A804D

EC50 mM (% max) 3.4 ⫾ 0.3 (149 ⫾ 2) 10.2 ⫾ 0.7 (157 ⫾ 9) N.D. N.D. N.D. N.D. N.D.

Receptors (1:1 ratio) BS-wt ⫹ wt BS-R227Q ⫹ wt BS-E297K ⫹ wt BS-C395R ⫹ wt BS-R795W ⫹ wt BS-P798T ⫹ wt BS-A804D ⫹ wt

EC50 mM (% max) 3.2 ⫾ 0.4 (130 ⫾ 2) 7.7 ⫾ 1.3 (125 ⫾ 1) 6.7 ⫾ 0.9 (127 ⫾ 1) 6.6 ⫾ 0.4 (129 ⫾ 2) 5.3 ⫾ 0.5 (136 ⫾ 3) 5.0 ⫾ 0.3 (150 ⫾ 2) 9.9 ⫾ 0.6 (122 ⫾ 2)

EC50 values were determined from data in Fig. 2. EC50 values are presented as mean ⫾ SE. Maximal ADIS responses (%max) are presented as mean ⫾ SE. N.D., Not defined.

with 2.5 ␮M Fura Red-AM with an equal volume of pluoronic and 2.5 mM probenecid in 0.5 mM Ca2⫹ bath for 30 min at 37 C/5% CO2. Cells were washed and incubated for an additional 30 min at room temperature before imaging using a 594-nm laser line with the angle set for wide-field acquisition. The cells were continuously superfused with 0.5 mM Ca2⫹ bath solution, or as indicated in the figure legends.

Fluorescence resonance energy transfer (FRET) by TIRFM Cells were transiently transfected with FLAG-CaSR-CFP and FLAG-BS-E297K at the indicated DNA ratios (total 1 ␮g, equalized with pcDNA3.1) and grown on 22-mm glass coverslips coated with human fibronectin. At 72 h after the transfection, cells were prepared for TIRFM as described. CFP and yellow fluorescent protein (YFP) channels were acquired with the

440-nm laser line, 470/22-nm emission filter or 514-nm laser line, 560/50-nm emission filter, respectively. The FRET channel consisted of the 440-nm laser line and 560/50-nm emission filter. TIRFM angles were individually adjusted for each excitation wavelength.

Image/data analysis TIRFM images were analyzed with ImageJ software (National Institutes of Health). The averaged mean surface intensities for regions of interest in individual cells were normalized to the first value in the time series. Background intensities were subtracted using the Subtract Background function in ImageJ. Normalized data from individual cells were averaged over independent experiments, analyzed, and plotted with SigmaPlot version 11 (San Jose, CA). Raw, normalized intensity data at each [Ca2⫹] was first fitted with a sigmoidal equation, Y ⫽ Imin ⫹ (Imax-Imin)/(1⫹10ˆ[(LogI50-X)ˆSlope]) to determine the maximum intensity value. The fitted maximum at each [Ca2⫹] was then fitted with a sigmoidal equation to extract EC50 values as Y ⫽ 100/(1⫹10ˆ[(LogEC50-X)ˆHillSlope]). Net FRET (nFRET) was calculated as nFRET ⫽ IFRET ⫺ (IYFP 䡠 A) ⫺ (ICFP 䡠 B), where IFRET, IYFP, and ICFP represent emission intensities from the respective channels. A and B represent bleed-through factors from the CFP and YFP channels, respectively. nFRET was normalized as FRETN (percentage) ⫽ nFRET/(ICFP 䡠 IYFP)1/2 䡠 100.

Results

FIG. 3. Ca2⫹-ADIS responses of FHH mutants. A, HEK293 cells were transfected with BS-CaSR-mutant constructs (R227Q, f; E297K, Œ; C395R, ; R795W, ⽧; P798T, E; or A804D, 䡺) or BS-wt (F). Experiments as in Fig. 1D were used to characterize the Ca2⫹dependence of ADIS of BS-mutants or BS-wt. Data were fitted to estimate EC50 and maximal responses (Table 1) and plotted as mean ⫾ SE (n ⫽4 cells). Inset symbols denote expression of BS-tagged homodimers. B, Western blot of lysates from HEK293 cells transiently expressing BS-wt or BS-mutants; the same blot was probed with both anti-CaSR antibody (upper portion) and anti-GAPDH antibody (lower portion, loading control).

We recently developed an experimental approach that allows simultaneous, real-time monitoring of plasma membrane levels of CaSR by TIRFM and measurement of intracellular Ca2⫹, using wide-field imaging of Fura Red (23). Imaging of plasma membrane-localized CaSR is optimized with a chimera of wild-type CaSR (BS-wt) having amino terminal additions of a minimal bungarotoxin binding site followed by a GFP variant, which is sensitive to pH, Super Ecliptic pHluorin (Fig. 1A). Fluorescence of intracellular BS-wt is minimized by the relatively acidic pH in intracellular organellar compartments, whereas plasma membrane-localized BS-wt fluorescence is enhanced by the slightly alkaline pH of the bath solution (23). Figure 1B shows representative TIRFM images from the experiment in Fig. 1C. HEK293 cells transfected with



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FIG. 4. Coexpression of BS-mutants with wild-type CaSR rescues ADIS. A, HEK293 cells expressing BS-FHH mutants (R227Q, f; E297K, Œ; C395R, ; R795W, ⽧; P798T, E; or A804D, 䡺) or BS-wt (F) were cotransfected with HA-wt at a 1:1 ratio of cDNAs. Experiments as in Fig. 1D were used to characterize the Ca2⫹ dependence of ADIS of BS-mutants or BS-wt. Data were fitted and plotted as described, mean ⫾ SE (n ⫽ 4 cells), EC50s, and maximal responses in Table 1. Inset symbols indicate BS-tag on mutant CaSRs (black), with silent CaSR-wt (white). B, Western blots of coimmunoprecipitations (with either anti-GFP to probe for BS-containing receptors or anti-HA antibodies) and corresponding lysates from cells transfected with BS-mutant plus HA-wt (1:1 ratio of cDNAs). Immunoprecipitations (IPs) were blotted with the partner antibody, and lysates were probed with anti-CaSR antibody. Blots are representative of three independent experiments. IB, Immunoblot. C, HEK293 cells transiently expressing 1:1 cDNA ratios of BS-E297K ⫹ wt or BS-wt ⫹ E297K (D) and loaded with Fura Red were imaged by TIRFM and wide-field microscopy to measure changes in plasma membrane Super Ecliptic pHluorin (SEP) and Fura Red fluorescence changes in response to 10 mM bath Ca2⫹. Images were collected in 15-sec intervals, and data were plotted as mean ⫾ SE (n ⫽ 4 cells). Inset symbols denote tracking of either BS-mutant (black) or BS-wt (white) receptors.

BS-wt were initially monitored in 0.5 mM, and then bath solution containing 10 mM extracellular Ca2⫹ was applied. Intracellular Ca2⫹ increased rapidly, whereas net plasma membrane BS-wt increased more slowly to a new steady state (Fig. 1C). Neither intracellular Ca2⫹ nor net receptor levels declined until the bath Ca2⫹ was returned to 0.5 mM. We have previously characterized this link between CaSR signaling and net plasma membrane levels of CaSR and shown that the net increase in plasma membrane CaSR is due to increased anterograde trafficking from secretory pathway compartments rather than a change in constitutive endocytosis (23). Both the rate of rise in plasma membrane CaSR abundance and the net steadystate level of plasma membrane BS-wt increase as a function of extracellular Ca2⫹. Figure 1D illustrates experiments in which bath Ca2⫹ was increased from 0.5 mM over the range from 1 to 30 mM. The Ca2⫹-response relation for net plasma membrane BS-wt is illustrated in Fig.

1E, with EC50 3.4 ⫾ 0.3 mM, which is comparable with the EC50 for activation of CaSR signaling in HEK293 cells, i.e. 3.3 mM (26). Results with BS-wt argue that measurement of steady state CaSR can serve as a surrogate for CaSR function, with the additional benefit of allowing explicit tracking of changes in plasma membrane-localized CaSR. This is particularly relevant for the study of CaSR mutations because we (12–14) and others (15, 16) have shown that a significant fraction of mutations causing FHH/NSHPT have defects in trafficking or targeting to the plasma membrane. The ability to track signalingmediated changes in plasma membrane CaSR allows us to determine the mechanism(s) contributing to signaling defects. The most severe phenotypes are seen in individuals bearing mutations in both CASR alleles. Figure 2 illustrates the locations of mutants in the CaSR domain structure and the signaling and ADIS responses of mutants previously shown to have altered trafficking/targeting to

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the plasma membrane, including R227Q, E297K, C395R, R795W, P798T, and A804D (14). For each mutant generated in the BS-wt background, we assessed the intracellular Ca2⫹ (Fura Red imaging) and net plasma membrane ADIS (TIRFM) responses to 30 mM extracellular Ca2⫹. For all mutants, there was a strong correlation between the intracellular Ca2⫹ and ADIS responses, with wt (Fig. 1C) approximately equal to A804D, greater than R227Q greater than P798T, greater than C395R, approximately equal to R795W, approximately equal to E297K (Fig. 2 and Table 1). Because many CaSR mutations cause a reduction in the efficacy of Ca2⫹ for activation of signaling, we determined the Ca2⫹ dose-response relations for the net increase in plasma membrane receptors. Experiments such as those illustrated in Fig. 1D were performed at various Ca2⫹ concentrations over the range from 1 to 30 mM for each mutant. The maximal steadystate increase in plasma membrane abundance at each [Ca2⫹] was fitted with the Hill equation for each mutant, illustrated in Fig. 3A. Derived fit parameters are listed in Table 1. Only BS-R227Q showed saturation of the insertional response, and therefore was fitted, with an EC50 of 10.2 mM, compared with 3.4 mM for BS-wt. BS-A804D showed significant increases in plasma membrane levels but did not reach saturation, even at 30 mM, whereas all other mutants showed only minor increases at the highest Ca2⫹ concentrations. We examined receptor expression levels in lysates by Western blotting (Fig. 3B). CaSR is expressed at the endoplasmic reticulum as a core glycosyated form of 140 kDa and matures in the Golgi to a 160-kDa form. The blot illustrates that the dominant form for wt and all


mutants is the immature, 140-kDa form. CaSR and all mutants are also processed, to varying extents, to the mature, 160-kDa form. We probed the same blot for GAPDH as a loading control. It is not possible to strictly correlate the abundance of the 160-kDa form observed on Western blots with ADIS because the ADIS measurement represents the normalized increase in plasma membrane CaSR evoked by extracellular Ca2⫹. However, the results of Figs. 2 and 3 confirm a severe reduction in Ca2⫹ sensitivity for both receptor signaling and agonist-evoked trafficking responses when mutants are expressed alone, i.e. they form only homodimers. Patients with a single mutated CASR allele have relatively benign symptoms (7), indicating significant rescue of function by the wild-type allele. The ability to track only receptor chimeras (BS-wt or BS-FHH mutant) by TIRFM allows us to directly assess the contributions of wild-type CaSR to mutant CaSR trafficking. To mimic the single allele mutation state in patients, we coexpressed BS-mutant plus HA-wt (1:1 ratio of cDNAs) and determined the Ca2⫹ dose-response relations for ADIS of BSmutants. For comparison in this experimental paradigm, we cotransfected BS-wt plus HA-wt (1:1 cDNA ratio), which reduced the maximal response compared with transfection of BS-wt alone (see Table 1). Figure 4A illustrates the results for ADIS of BS-wt (dashed line) and all BS-mutants (solid lines) coexpressed with HA-wt. All of the mutants showed a significant leftward shift in Ca2⫹ADIS response relations compared with the expression of mutant alone (recall Fig. 3A). As indicated in Table 1, most mutants had EC50s for the insertional response in the range of 5–10 mM Ca2⫹, with maximal responses

FIG. 5. FRET confirmation of wild-type CaSR and BS-E297K heterodimerization. A, HEK293 cells transfected with wild-type FLAG-CaSR-CFP ⫹ BS-E297K at different cDNA ratios as indicated were imaged by TIRFM. Representative images from the CFP, YFP, and nFRET channels are presented. Images are representative of 68 cells. B, nFRET values from multiple experiments were transformed to FRETN (percentage) as described in Materials and Methods for each cDNA expression ratio. Data were plotted as mean ⫾ SE (n ⫽ 85 cells at each ratio), compared with 1:1 ratio by ANOVA, post hoc Dunnett’s (*, P ⬍ 0.05). Inset symbols indicate predicted distribution of dimeric forms, wt-CFP (blue), BS-E297K (yellow).



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imaging and immunoprecipitated with either anti-GFP antibody to pull down/ detect BS-mutants or anti-HA antibody to pull down/detect HA-wt. Blots were probed with the partner-specific antibody. Lysates were probed with anti-CaSR antibody (Fig. 4B) and showed all forms of CaSR, i.e. multiple bands are due to differential masses contributed by epitope and chimeric tags. Results confirm heterodimerization of all mutants with HA-wt. Finally, we determined whether the Ca2⫹ signaling and ADIS responses were comparable when either the BS-wt or mutant receptor was tracked by TIRFM. For these experiments, we chose a mutant, E297K, which has minimal Ca2⫹ signaling and plasma membrane targeting when expressed as a homodimer (Fig. 2 and Table 1). Figure 4C shows averaged responses when BS-E297K was monitored in the presence of HA-wt, and Fig. 4D shows similar experiments when BS-wt was monitored in the presence of E297K. In both cases, signaling was comparable, with a rapid initial response to 10 mM Ca2⫹ due to activation of CaSR-wt. The insertional response of BS-wt (Fig. 4D) was significantly higher than the response of BS-E297K (Fig. 4C) because both BS-wt homo- and heterodimers are monitored in this configuration of the assay. Overall, the results of Fig. 4 argue that hetFIG. 6. ADIS and Ca2⫹ signaling of BS-E297K are regulated CaSR-wt. HEK293 cells were transfected at cDNA ratios of CaSR-wt to BS-E297K of 1:1; 1:4, and 4:1. A, Ca2⫹-ADIS erodimerization with wild-type CaSR response relations, determined as in Fig. 1D, for control cells expressing BS-wt ⫹ wt (1:1, F); and Ca2⫹ signaling by wild-type CaSR or cells expressing wt ⫹ BS-E297K (1:1, f; 4:1, Œ; or 1:4, 䡺). Data were plotted as mean combine to increase the trafficking of ⫾ SE (n ⫽ 4 cells). EC50s and maximum responses are provided in Table 2. Inset indicates tracking of E297K (black). B–D, Fura Red responses of cells expressing CaSR-wt ⫹ BS-E297K mutant receptors to the plasma 1:1 (B), 4:1 (C), and 1:4 (D) cDNA ratios, exposed to the indicated Ca2⫹ bath solutions. Inset membrane. symbols indicate predicted distribution of dimeric forms, wt (white) and BS-E297K (black). E, We next determined the relative 2⫹ Ca -ADIS response relations for cells expressing E297K ⫹ BS-wt (Œ) or wt ⫹ BS-wt (F) at contributions of wild-type CaSR Ca2⫹ 4:1 ratios. These are plotted as mean ⫾ SE (n ⫽ 4 cells), EC50s, and maximum responses as in Table 2. Inset symbols denote tracking of BS-wt (white). F, Fura Red responses for cells signaling and heterodimerization to expressing CaSR-wt ⫹ BS-wt (4:1 ratio), as described in B–D. G, Fura Red responses for cells the rescue of FHH/NSHPT mutant expressing E297K ⫹ BS-wt (4:1 ratio), as described in B–D. CaSR targeting to the plasma membrane. We hypothesized that biasing comparable with or greater than BS-wt. These studies assembly toward heterodimerization would afford the argue that signaling by HA-wt and/or heterodimerization greatest mutant trafficking rescue if a CaSR-wt partner is with HA-wt drives plasma membrane increases of mutant required to overcome the trafficking block. However, if CaSRs. To confirm expression and heterodimerization of Ca2⫹ signaling dominates, we expect promoting assemmutant with HA-wt, cells were transfected as for TIRFM bly of CaSR-wt homodimers would afford the greatest

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rescue. We transfected different ratios of CaSR-wt to BSE297K cDNAs and confirmed that this biasing strategy resulted in differential assembly of heterodimers using FRET. HEK293 cells were transfected with CaSR-CFP plus BS-E297K at 1:1, 4:1, and 1:4 ratios, and Fig. 5A shows images from the CFP, YFP, and nFRET channels. Figure 5B illustrates the calculated normalized FRETN (%) for multiple experiments, with a cartoon illustrating the predicted dimer forms. At a 1:1 ratio, heterodimer formation is favored, leading to significant FRETN (%), whereas at a 4:1 ratio of CaSR-CFP to BS-E297K, homodimerization of CaSR-CFP predominates, leading to a significant reduction in FRETN (%). A ratio of 1:4 favors heterodimerization, leading to a FRETN (%) limited primarily by the availability of CaSR-CFP. Figure 5 demonstrates that expression of different ratios of CaSR-CFP to BS-E297K leads to the predicted formation of homo- and heterodimers. We next used the ratios of CaSR-wt and BS-E297K characterized in Fig. 5, which bias toward heterodimerization, to determine the effects on ADIS and Ca2⫹ signaling responses. Figure 6A illustrates ADIS of BS-E297K for expressed ratios of wt to BS-E297K of 1:1, 1:4, and 4:1 (solid lines). Control experiments having 1:1 cDNA ratio of BS-wt to wt are plotted for comparison (dashed line). Fits for all data are listed in Table 2. Also illustrated in Fig. 6, B–D, are the corresponding intracellular Ca2⫹ responses in representative cells expressing the indicated cDNA ratios. There is a strong correlation between the degree of rescue of ADIS and the elicited Ca2⫹ signaling responses, i.e. the 4:1 ratio of wt to BS-E297K leads to near wild-type ADIS and signaling (Fig. 6A, black triangles, and Fig. 6C), whereas neither ADIS nor Ca2⫹ signaling responses of the 1:4 ratio saturate at 30 mM (Fig. 6A, open squares, and Fig. 6D). These experiments argue for a dominant role for signaling in driving plasma membrane trafficking of FHH/NSHPT mutants. To further test the idea that Ca2⫹ signaling dominates in the control of CaSR trafficking, we determined the impact of coexpression of BS-wt (1:4 ratio to favor heterodimerization) TABLE 2. Ca2⫹-ADIS relations for FHH mutants coexpressed with wild-type CaSR Receptors (ratio) wt ⫹ BS-wt (1:1) wt ⫹ BS-E297K (1:1) wt ⫹ BS-E297K (4:1) wt ⫹ BS-E297K (1:4) wt ⫹ BS-wt (4:1) E297K ⫹ BS-wt (4:1)

EC50 mM 3.2 ⫾ 0.6 6.6 ⫾ 0.4 4.8 ⫾ 0.4 N.D. 2.5 ⫾ 1.1 7.4 ⫾ 0.7

HEK293 cells were transfected with the indicated ratios of wild-type and E297K constructs. EC50 values were determined from data in Fig. 6, A and E, and are presented as mean ⫾ SE. N.D., Not defined.


with the signaling-defective mutant E297K. As a control, we coexpressed BS-wt plus wt at a 1:4 cDNA ratio. The Ca2⫹ signaling responses are illustrated in Fig. 6, F and G. The control experiment indicates robust Ca2⫹ signaling, whereas coexpression of BS-wt plus E297K shows significantly reduced Ca2⫹ signaling, as expected from a bias toward BS-wt:E297K heterodimers. Recall from Fig. 2A that homodimers of E297K undergo little ADIS, even at 30 mM Ca2⫹. Figure 6E illustrates the Ca2⫹-activated insertional response of BS-wt in heterodimers with E297K compared with BS-wt with wt (dotted line). There is a significant rightward shift in the response, with EC50 approximately 7.4 mM. Both the Ca2⫹ signaling and insertional responses are comparable with those seen when BS-E297K and wt were expressed at 1:1 ratios, again fostering heterodimerization. These results argue that signaling is critical to the insertional response and that partial function of heterodimers plus signaling by CaSR-wt homodimers contribute to rescue of trafficking-defective FHH/NSHPT mutants to the plasma membrane. Previous studies have shown rescue of FHH mutant signaling upon coexpression of two mutants having defects in distinct receptor domains, in particular, receptors having point mutations in the ECD plus CaSR having a large carboxyl terminal deletion (27). We tested for rescue of ADIS and Ca2⫹ signaling for E297K with the CaSR⌬868 truncation (1:1 ratio of cDNAs), tracking either BS-⌬868 (Fig. 7, A and C) or BS-E297K (Figs. 7, B and D). When expressed alone, neither BS-⌬868 (Fig. 7A) nor BS-E297K (Fig. 7B) showed robust ADIS or Ca2⫹ signaling, even at 30 mM extracellular Ca2⫹. In contrast, when coexpressed, there was a significant restoration of Ca2⫹ signaling and ADIS, when tracking either BS-⌬868 (Fig. 7C) or BS-E297K (Fig. 7D). These results suggest the potential for functional complementation across the heterodimer, although it should be noted that ⌬868 represents wild-type CaSR through both the ECD and heptahelical domains. To further test heterodimer complementation, full-length mutants in distinct domains were coexpressed. We used the i3 loop mutant, R795W, plus several ECD mutants, G143E and E297K, compared with CaSR-wt. As expected, coexpression of BS-R795W ⫹ R795W shows minimal ADIS and Ca2⫹ signaling (Fig. 8A), and coexpression of BS-R795W ⫹ wt rescued both activities (Fig. 8B). In contrast, coexpression of BSR795W with either G143E (Fig. 8C) or E297K (Fig. 8D) did not rescue either ADIS or Ca2⫹ signaling. At least for these mutants, complementation of function did not occur across the heterodimer interface, arguing that significant rescue of trafficking requires Ca2⫹ signaling.



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mutants in the absence or presence of CaSR-wt while simultaneously monitoring intracellular Ca2⫹ as a reflection of net CaSR signaling (23). We examined a selection of FHH mutants that have previously been shown to have signaling and/or trafficking defects (14). Expression of the FHH mutants alone recapitulates the NSHPT phenotype, i.e. both ADIS and Ca2⫹ signaling in HEK293 cells are severely compromised, even at significantly elevated extracellular Ca2⫹. Only R227Q and A804D mediated ADIS and Ca2⫹ signaling, albeit at higher extracellular Ca2⫹ concentrations than CaSR-wt. When expressed in a 1:1 ratio with CaSR-wt, a condition that replicates the heterozygous condition leading to FIG. 7. Coexpression of E297K and CaSR⌬868 restores ADIS. HEK293 cells expressing 1:1 FHH, we observed rescue of Ca2⫹ sigcDNA ratios of BS-⌬868 ⫹ ⌬868 (A), BS-E297K ⫹ E297K (B), BS-⌬868 ⫹ E297K (C), or BSnaling, and more importantly, obE297K ⫹ ⌬868 (D) were loaded with Fura Red and imaged as described in Fig. 1C. Cells were exposed to 30 mM Ca2⫹ as indicated. ADIS responses are plotted as mean ⫾ SE (n ⫽ 4 cells), served trafficking of all FHH mutants and Fura Red responses are plotted for individual cells. to the plasma membrane. ADIS of mutants reached maximal levels comparaDiscussion ble with CaSR-wt, with EC50s in the range from 5 to 10 In this report, we used a high-resolution imaging tech- mM Ca2⫹. This is a fundamentally important result that nique to explore the agonist-driven trafficking of FHH argues that signaling is an absolute requirement for trafficking of CaSR to the plasma membrane. Furthermore, FHH mutants that are functionally inactive when expressed alone can be released to the secretory pathway by CaSR-wt signaling. These results argue that the benign phenotype that accompanies coexpression of FHH mutants with CaSR-wt results from Ca2⫹ signaling, which increases FHH mutant access to the plasma membrane. To fully explore the relative contributions of CaSR-wt signaling vs. heterodimerization to trafficking rescue of FHH mutants, we focused on the FHH mutant E297K. E297K is weakly targeted to the plasma membrane in the absence of CaSR-wt and elicits minimal Ca2⫹ signaling responses at 30 mM extracellular Ca2⫹. Coexpression with CaSR-wt, however, rescues ADIS of FIG. 8. Lack of complementation for trafficking of domain-specific CaSR mutants. A, BSR795W was coexpressed with R795W (1:1 cDNA ratio), and ADIS and Ca2⫹ signaling were E297K. Net Ca2⫹ signaling responses assessed as described in Fig. 1C, plotted as average ⫾ SE (n ⫽ 4 cells). B–D, Experiments as in in this experimental paradigm cannot A to determine ADIS and Ca2⫹ signaling responses upon coexpression (1:1 cDNA ratio) of be ascribed to homodimers vs. hetBS-R795W with CaSR-wt (B), FLAG-G143E (C), and FLAG-E297K (D). ADIS responses are plotted as average ⫾ SE (n ⫽ 4 cells), and Fura Red responses are plotted for individual cells. erodimers of mutant and/or CaSR-wt.

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We therefore used a biasing strategy based on coexpression of ratios of cDNAs to drive assembly of heterodimers, as confirmed by FRET analysis. Rescue of E297K trafficking correlated with net Ca2⫹ signaling. Expression ratios that promoted strong intracellular Ca2⫹ responses showed the most significant rescue, whereas a ratio that elicited Ca2⫹ oscillations up to 30 mM extracellular Ca2⫹ showed the weakest rescue of E297K trafficking. Similarly, coexpression of CaSR-wt with excess E297K led to intracellular Ca2⫹ oscillations over the entire range of extracellular Ca2⫹ and compromised trafficking of CaSR-wt. Overall, these studies suggest that rescue of FHH mutant trafficking is ultimately dependent on activation of Ca2⫹ signaling responses that reach beyond the threshold for intracellular Ca2⫹ oscillations, i.e. rescue relies on the summation of CaSR-wt homodimer plus CaSR-wt/mutant heterodimer signaling. Complementation of function across the dimer interface has been described for members of GPCR family C (28). For ␥-aminobutyric acid type B receptors, complementation between two subunits is required for wild-type receptor function, with each partner making specific contributions to dimer activation (29). Functional complementation for CaSR has been described for select mutations identified in patients (27) and requires mutations in distinct domains, e.g. in the ECD plus carboxyl terminus. In particular, strong complementation was observed for coexpression of ECD mutants with A877STOP, a mutant having insertion of an Alu sequence at the codon for A877, leading to deletion of most of the CaSR carboxyl terminus (27, 30). In the present study, we observed restoration of ADIS and Ca2⫹ signaling when mutants were coexpressed with the CaSR truncation ⌬868 but not when they were coexpressed with full-length R795W. The difference is particularly striking for the E297K mutant, which cannot be rescued by R795W (i3 loop mutant) but is fully rescued by CaSR⌬868 (carboxyl terminal truncation). We note, however, that we have recently shown that activation of CaSR⌬868 elicits a very weak ADIS response (23), primarily because loss of the carboxyl terminus removes critical protein interaction site(s) that mediate ER retention (24). In particular, the 14-3-3 binding site is localized to residues 891– 898 (31) and contributes to the signaling-regulated ADIS response (23). Coexpression of CaSR⌬868 with a receptor having an intact carboxyl terminus restores regulated retention (31), and thus, it is possible that restoration of ADIS and Ca2⫹ signaling is a result of signaling by CaSR-⌬868 alone. These studies do not rule out the potential for complementation across the CaSR dimer interface for specific combinations of mutations. They do suggest, however, that the functional complementation observed


when an FHH mutant is coexpressed with truncated wildtype CaSR is due to net Ca2⫹ signaling mediated by the wild-type CaSR ECD plus heptahelical domains. Results of this study suggest that CaSR harboring FHH/NSHPT mutations can be driven to the plasma membrane by CaSR signaling. The ADIS mechanism requires a threshold level of plasma membrane-localized CaSR at low extracellular Ca2⫹ to permit a robust intracellular Ca2⫹ response when extracellular Ca2⫹ is elevated. Although the FHH/NSHPT mutants studied in this report showed some plasma membrane localization at 0.5 mM Ca2⫹, many were incapable of eliciting a robust Ca2⫹ signaling response when challenged with elevated extracellular Ca2⫹; thus, ADIS was not induced. Coexpression with CaSR-wt, however, allowed sufficient activation of signaling that FHH/NSHPT mutants showed an ADIS response. The present studies offer insights into the differential impact of FHH/NSHPT mutations in various tissues expressing CaSR. Cells/tissues expressing other Gq-coupled GPCRs capable of eliciting sustained increases in intracellular Ca2⫹, rather than a rapidly desensitizing response, may show partial rescue of CaSR-mutant trafficking and signaling, moderating tissue phenotype. In this scenario, the greatest impact of FHH/ NSHPT mutations will be in cells/tissues in which sustained intracellular Ca2⫹ responses are uniquely generated by CaSR. From a clinical perspective, the present results argue that any strategy that increases CaSR signaling will result in a feed-forward potentiation of further signaling, facilitating a net increase in both CaSR-wt and FHH/NSHPT mutant abundance at the plasma membrane. Recent studies have argued that membrane-permeant calcimimetics foster enhanced plasma membrane targeting and signaling of FHH/NSHPT mutants by acting as pharmacochaperones and promoting proper folding at the ER and/or biasing receptor conformations to promote release from the ER (12, 32). The present results propose an additional role for calcimimetics, i.e. potentiation of plasma membrane signaling to increase CaSR FHH/NSHPT mutant trafficking to the plasma membrane.

Acknowledgments We thank Dr. Mingliang Zhang and Elissa White for the generation of some of the constructs and members of the Breitwieser laboratory for helpful discussions. Address all correspondence and requests for reprints to: Gerda E. Breitwieser, Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, Pennsylvania 17822-2604. E-mail: [email protected].


This work was supported by Great Rivers Affiliate AHA Postdoctoral Fellowship (to M.P.G.) and Geisinger Clinic (to G.E.B.). Disclosure Summary: The authors have no conflicts of interest to report.


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