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Endocrinology Vol. 145, No. 8 3892-3903
Copyright © 2004 by The Endocrine Society

The Role of Cysteines and Charged Amino Acids in Extracellular Loops of the Human Ca2+ Receptor in Cell Surface Expression and Receptor Activation Processes

Kausik Ray, Sanchita P. Ghosh and John K. Northup

Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Kausik Ray, Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, 5 Research Court, Room 2A11, Rockville, Maryland 20850. E-mail: rayk{at}nidcd.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ca2+ receptor is a plasma-membrane bound G protein-coupled receptor stimulated by extracellular calcium [Ca2+]o and other di- and poly-cations. We investigated the role in receptor activation of all the charged amino acid residues and cysteines in the three extracellular loops (EL1, 2, and 3) of the human Ca2+ receptor by alanine-scanning mutagenesis. The mutant receptors were transiently expressed in HEK-293 cells, and cell surface expression patterns were analyzed by endoglycosidase-H digestion, immunoblotting, intact cell ELISA, and hydrolysis of phosphoinositides (PI) induced by [Ca2+]o. The mutation of Cys677 and Cys765 located in EL1 and EL2, respectively, ablated PI hydrolysis completely, showed less than 5% cell surface expression of the wild-type receptor, and were not properly glycosylated. Replacement of the charged residues by using a single mutation or multiple alanine mutations in EL1, 2, and 3 produced only minor changes in receptor activation, except for Glu767 and Lys831. The E767A and K831A mutations in EL2 and EL3, respectively, showed gain-of-function by significantly enhancing apparent [Ca2+]o affinity. E767A and K831A exhibited EC50 values of 2.1 and 2.8 mM, respectively, for [Ca2+]o-stimulated PI hydrolysis as opposed to EC50 value of 4.2 mM for the wild-type receptor. Like E767A, substitutions of Glu767 with Gln and Lys was similarly activating, whereas Asp substitution displayed wild-type [Ca2+]o sensitivity. Substitution of Lys831 with Glu but not with Gln showed similar activating effect as Ala replacement. A double-mutant E767K/K831E in which charged residues were switched positions showed impaired cell surface expression and failed to respond to [Ca2+]o. Taken together, these results suggest that in ELs, two cysteines form critical disulfide links, and the side chains of Glu767 and Lys831 are probably involved in ionic interactions with other prospective oppositely charged residues. Some of these interactions could be important for receptor folding and also may contribute to keep the Ca2+ receptor transmembrane helix bundle in an inactive conformation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN CALCIUM receptor (hCaR) is the member of the G protein-coupled receptor (GPCR) superfamily that regulates extracellular calcium [Ca2+]o ion homeostasis by controlling the rate of PTH secretion from the parathyroid gland and the rate of calcium reabsorption by the kidney. A number of naturally occurring inactivating and activating mutations of the hCaR have been identified in subjects with disorders of [Ca2+]o homeostasis; familial benign hypocalciuric hypercalcemia and autosomal dominant hypocalcemia/sporadic hypocalcemia, respectively (1).

Most of the structural information for GPCRs to date have been obtained from the largest family (family 1) rhodopsin-like GPCRs and focused on mutational analyses on the seven transmembrane (7TM) helices for locating amino acids that are specifically involved in ligand binding and mechanisms of receptor activation (2). However, there is increasing evidence for diverse modes of ligand binding, signal generation, and 7TM transduction within GPCR subfamilies (3, 4). The hCaR belongs to the structurally unique family 3 characterized by very large amino-terminal extracellular domains (ECDs) that includes metabotropic glutamate receptors (mGluR1–8), putative pheromone receptors of the vomeronasal organ, sweet taste receptors 1–3, and {gamma}-aminobutyric acidB receptors. Studies of chimeric receptors containing hCaR ECD and mGluR1 7TM domains have confirmed that hCaR ECD confers responsiveness to [Ca2+]o on the chimeric receptors (5, 6). This defines a ligand-binding role similar to that defined for the ECD structures of mGluR1 and other family 3 receptors (7). The three-dimensional structure of the mGluR1 ECD determined by x-ray crystallography precisely defines the glutamate binding site within the ECD and suggests that a closed conformation of the ECD is the active state (8). Interestingly, unlike mGluR1, hCaR seems to have Ca2+ binding sites both in the ECD and the 7TM domain. An hCaR mutant 7TM construct lacking the ECD (T903-Rhoc) responds to [Ca2+]o and other di- and poly-cations (5, 6, 9). Furthermore, (R)-N-(3-methoxy-{alpha}-phenylethyl)-3-(2'-chlorophenyl)-1-propylamine hydrochloride (NPS-R568), an allosteric calcimimetic compound, synergistically enhances these cationic responses (5, 6, 9).

An acidic residue (Glu837) located in extracellular loop (EL) 3 of the 7TM has been reported to interact with NPS-R568 but might not be involved in Ca2+ binding (10). We had previously hypothesized that a cluster of acidic residues in the EL2 might be involved directly in Ca2+-binding in the 7TM core, but our initial results suggested, rather, that these are probably involved in the allosteric regulation of the ECD-deleted hCaR, T903-Rhoc (9). Thus, despite a low sequence identity with rhodopsin-like GPCRs, the hCaR seems to have a ligand-binding pocket formed by its 7TM segments. Moreover, it has been speculated that an allosteric interaction of the ECD and movement of 7TM helices might participate in the activation of the hCaR and other family 3 GPCRs (4, 11). Such ECD-7TM interactions might be mediated through the three ELs connecting the 7TM helices because they are also critical for many GPCR activation and ligand binding processes (12). One key feature exhibited by many GPCRs is the presence of a pair of conserved Cys residues in EL1 and EL2 that forms a disulfide bridge and constrains the 7TM helical bundle (12). Substitution of the conserved Cys residues in several GPCRs abolishes ligand binding by either enabling signaling-defective conformations of the mutant receptors or impairing cell surface receptor expression altogether (12, 13, 14, 15). Other interactions such as ionic or salt bridges between charged residues have also been shown to constrain the 7TM helical bundles (12, 13, 14, 15).

In this present study, we sought to identify amino acids that are involved either directly in Ca2+ binding or in conformational restriction of the hCaR. We hypothesized that substitution of either of the two conserved cysteines forming a potential disulfide bond or the charged amino acids possibly involved in ionic interactions (Arg, Lys, Asp, or Glu) within the ELs of the hCaR 7TM structure might have marked effects on folding/processing or [Ca2+]o-induced activation processes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Site-directed mutagenesis of the hCaR
Site-directed mutagenesis was performed on hCaR cDNA in the pCR3.1 vector (16) using a commercial kit (QuikChange site-directed mutagenesis kit, Stratagene Inc., La Jolla, CA), according to the manufacturer’s instructions as described previously (17). Briefly, a pair of complementary primers with 30–35 bases was designed for each mutation with targeted residue placed in the middle of the primers (a list of these primer sequences will be provided upon request). The parental hCaR cDNA in the pCR3.1 vector was amplified using Pyrococcus furiosus DNA polymerase with these primers for 12 cycles in a DNA thermal cycler. After digestion of the parental DNA with DpnI, the newly synthesized DNA with the nucleotide substitution was transformed into Escherichia coli (DH-5{alpha} strain). The double and multiple mutants were created by using single mutant cDNA as template in the subsequent round of mutagenesis. The mutations were confirmed by automated DNA sequencing using a Taq DyeDeoxy terminator cycle sequencing kit and ABI prism-377 DNA sequencer (Applied Biosystems, Foster City, CA). A single positive clone of individual mutant construct for a mutation was tested for functional response and compared with the wild-type hCaR activity. For each mutant receptor construct that showed noticeable differences in functional activities compared with wild-type hCaR, we analyzed at least two independent clones for individual mutation and confirmed that they had identical functional properties. We analyzed four independent clones of C677A, C765A, and E767K/K831E; three independent clones each of E767A; and two independent clones each of E/D671,674A, R680A, E767A, E3A, E/D5A, E767D, E767K, E767Q, K831A, K831E, and K831Q. Moreover, we completely sequenced the mutant cDNAs of E767A and K831A to confirm the absence of mutations in other regions in these mutant constructs. In addition, three independent clones each of D758A or E759A were tested in functional assays because our results differed from an earlier reported study (10).

Transient transfection of wild-type and mutant receptors in HEK-293 cells
For transfection, plasmid DNA was diluted in DMEM (BioFluids Inc., Rockville, MD), mixed with diluted Lipofectamine (Invitrogen, Carlsbad, CA), and the mixture incubated at room temperature for 30 min. The DNA-Lipofectamine complex was further diluted in serum-free DMEM, and 8 µg DNA was added to 80–90% confluent human embryonic kidney (HEK)-293 cells plated in 75-cm2 flasks. This amount of DNA was determined to generate optimal transfection efficiency and highest expression level of the hCaR. After 5 h of incubation, an equal volume of DMEM containing 20% fetal bovine serum (FBS) (BioFluids) was added, and the media were replaced 24 h after transfection with complete DMEM containing10% FBS. Membrane protein extraction for immunoblotting, and functional phosphoinositide (PI) hydrolysis assays were performed 48 h after transfection.

Immunoblot analysis of receptor expression
Confluent cells in 75-cm2 flasks were rinsed with ice-cold PBS and scraped on ice in solution A [5 mM Tris-HCl (pH 7.5), 2 mM EDTA,10 mM iodoacetamide] with freshly added Complete protease inhibitors cocktail (Roche Applied Science, Indianapolis IN). Cells were forced through a 22-gauge needle five to eight times, and the crude membranes were pelleted by centrifugation at 15,000 rpm for 10 min at 4 C. The pellet was resuspended and solubilized in solution B containing 20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, and 1% TritonX-100 with freshly added protease inhibitors cocktail. The protein content of each sample was determined by the modified Bradford method (Bio-Rad Laboratories, Hercules, CA), and approximately 18 µg of protein per lane was separated on 6% sodium dodecyl sulfate-polyacrylamide gels (Invitrogen). The gel was first fixed in 1 x transfer buffer (Tris-glycine) containing 20% methanol, and then the proteins on the gel were electrotransferred to nitrocellulose membrane and incubated with the hCaR-specific monoclonal antibody 6D4 (kindly provided by Dr. Tyler Miller, Case Western University, Cleveland, OH) described earlier (18). Antibody absorption was detected by a secondary goat antimouse antibody conjugated to horseradish peroxidase (Kierkegaard and Perry Laboratories, Gaithersburg, MD), and immunoreactivity was detected with an enhanced chemiluminescence system (Amersham Corp., Arlington Heights, IL).

Endoglycosidase-H (Endo-H) treatment of detergent-solubilized extracts
For cleavage with Endo-H, detergent-solubilized membrane extracts (approximately 40 µg protein) in solution B were incubated with 0.5 mU Endo-H for 1 h at 30 C before loading approximately 18 µg solubilized protein samples on 6% sodium dodecyl sulfate-polyacrylamide gels as described above for immunoblotting.

Intact cell enzyme-linked immunoassay of hCaR cell surface expression
Mutant receptors or wild-type hCaR was transiently expressed in HEK-293 cells. Forty-eight hours after transfection in either 75-cm2 flasks as described above or six-well plates (with 1 µg DNA per well), confluent transfected HEK-293 cells were detached with 1 mM EDTA in PBS containing 0.5% BSA. Cells were incubated with 0.5 ml of DMEM containing 10% FBS and 6D4 monoclonal anti-hCaR antibody at 4 C for 2 h. After three washes in PBS, cells were incubated similarly for 1 h with peroxidase-conjugated goat antimouse IgG{gamma} (Kirkegaard & Perry Laboratory). Peroxidase substrate [2.5 mM each of H2O2 and o-phenylenediamine in 0.1 M phosphate-citrate buffer, pH 5.0)] was added at room temperature after three washes, and the color reaction was measured at 525 nm in a spectrophotometer.

PI hydrolysis assay
PI hydrolysis in intact HEK293 cells transiently expressing hCaR was determined as previously described (16). Briefly, 24 h after transfection, cells from a confluent 75-cm2 flask were replated in a 24-well plate in medium containing 3.0 µCi/ml of 3H-myoinositol (NEN Life Science Products, Beverly, MA) in complete DMEM and incubated for another 24 h. The medium was then replaced with solution C [120 mM NaCl, 0.5 mM CaCl2, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl2, 20 mM LiCl in 25 mM 1,4-piperazinedietthanesulfonic acid (pH 7.2)], and the cells were incubated for 30 min before addition of test agents. After 45 min with the test agents, the PI hydrolysis was terminated by addition of 1 ml acid-methanol (1:1000 vol/vol) per well. Total inositol phosphates were fractionated by chromatography on 1 ml Dowex 1-X8 columns.

Data analysis
The [Ca2+]o saturation of PI hydrolysis responses were analyzed using GraphPad Prizm (version 3, GraphPad Software, San Diego, CA). Data fits for single-site and other ligand-binding models were compared for minimum residual errors. The coefficients for the best-fit models for each individual experiment were used to compute EC50 and Hill coefficient numbers reported in Tables 1–3GoGoGo. For all mutant receptors except E/D5A, the data were best fit by a Hill equation. For E/D5A the data were best fit to a single site, noncooperative model.


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  		TABLE 1. Exo-loop1 mutants: EC50 values and maximal PI response of mutant receptors

 

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  		TABLE 2. Exo-loop2 mutants: EC50 values and maximal PI response of mutant receptors

 

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  		TABLE 3. Exo-loop3 mutants: EC50 values and maximal PI response of mutant receptors

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The three ELs within the 7TM domain of the hCaR contain two conserved cysteines, four basic residues (Arg, Lys), and eight acidic residues (Asp, Glu) as depicted in Fig 1Go. Site-directed mutagenesis was performed to change these residues individually and in some cases collectively to alanines to assess the contributions of these residues to hCaR signaling.



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  		FIG. 1. Schematic representation of the amino acids within EL1–3 of hCaR. Only the 7TM domain residues from Gly613 to Lys863 are depicted here. The location of two cysteines (gray), four basic amino acids (yellow), and eight acidic residues (orange) in the EL1–3 are highlighted by the indicated colors. TM1-TM7 represent the 7TM helices of the hCaR.

 
Expression patterns and functional properties of EL1 mutant hCaRs
We first created a set of five alanine-substituted single-site mutants in which a single cysteine, two basic, and two acidic residues in EL1 were altered in the full-length hCaR cDNA construct. These mutants were as follows: E671A, D674A, C677A, R678A, and R680A. The analysis of the expression pattern of the wild-type hCaR and these five mutants in HEK293 cells is shown in Fig. 2Go. Under reducing conditions hCaR-specific 6D4 antibody detected two major monomeric bands in wild-type hCaR-expressing cells of approximately 150- and 130-kDa mass. Previously reported studies have shown that the monomeric 150-kDa band contains hCaR forms expressed at the cell surface and modified with N-linked, complex carbohydrates resistant to Endo-H digestion and the 130-kDa band contains high mannose-modified forms, trapped intracellularly and sensitive to Endo-H digestion (17, 19). We found that when an identical optimized amount of plasmid DNA (8 µg) was used for transient transfection of each construct in HEK 293 cells, except for C677A, all single mutant receptors expressed both the 150- and 130-kDa bands similar to the wild-type hCaR as seen by immunoblot detection (Fig. 2Go). The intensity of the cell surface expressed 150-kDa band for most mutants was similar to the wild-type hCaR, and Endo-H digestion minimally reduced the abundance of the 150-kDa band, whereas reducing the size of the 130-kDa band completely and generated a nonglycosylated band at around 120 kDa. C677A instead showed a single 130-kDa band, which was completely digested by Endo-H, indicating that these are mostly intracellular-trapped receptor forms. Also, note that whereas the presence of Endo-H-resistant 150-kDa band confirmed that a mutant receptor is expressed at the cell surface, because 150 kDa forms are modified by highly complex carbohydrates, this band showed somewhat variable intensities in different immunoblotting experiments. Therefore, comparison of 150-kDa band intensities may not be an accurate way to quantify the levels of cell surface expressed receptors.



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  		FIG. 2. Cell surface expression of EL1 mutants of the hCaR. Eighteen micrograms of crude membrane extract per lane obtained from HEK-293 cells transiently expressing wild-type (WT) hCaR or EL1 mutant receptors E671A, D674A, C677A, R678A, R680A, and E/D671,674A were incubated without (–) or with (+) Endo-H and resolved by SDS PAGE for immunoblotting with 6D4 antibody as described in Materials and Methods. The migration of the 150- and 130-kDa forms of the hCaR and the Endo-H-digested nonglycosylated band migration is shown as an asterisk on the right. This blot is representative of similar immunoblots from several different transfections.

 
Next, under similar transfection conditions, we determined the signaling properties of these mutants for [Ca2+]o-stimulated PI hydrolysis in intact cells. As shown in Fig. 3AGo, the point mutation of Glu671 or Asp674 to Ala did not alter the maximal responses of these mutants and the EC50 value of E671A was similar, and D674A was somewhat higher, compared with wild-type hCaR (Table 1Go). Two basic residue single mutants, R678A and R680A, showed very similar EC50 for PI response as the wild-type hCaR (Fig. 3BGo and Table 1Go). Whereas R680A showed a reduced maximal response to [Ca2+]o that was about 70–75% of the maximal wild-type hCaR response, it nevertheless showed very similar [Ca2+]o saturation like the wild-type hCaR. Whereas C677A showed no [Ca2+]o-dependent response in PI assay and immunoblot, data indicated this loss of function is probably due to lack of cell surface expression. Although the single point mutation of Glu671 and Asp674 did not significantly change receptor activity, we suspected both of these closely located acidic residues may contribute to a putative Ca2+ binding in 7TM helices.



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  		FIG. 3. [Ca2+]o saturation of PI hydrolysis in HEK-293 cells expressing wild-type (WT) hCaR or EL1 mutant hCaR. A, Transient expression of EL1 acidic residue mutants in HEK293 cells was accomplished by transfection with cDNAs encoding the WT ({blacktriangledown}), E671A ({triangleup}), D674A ({square}), or E/D671,674A ({bullet}) mutant receptors, and the measurement of PI hydrolysis in response to the indicated concentrations of Ca2+ was performed as described in Materials and Methods. B, HEK293 cells were transfected with cDNAs encoding WT ({blacktriangleup}), C677A ({triangledown}), R678A ({circ}), and R680A ({blacksquare}) and PI hydrolysis in response to the indicated concentrations of Ca2+ was performed as described in Materials and Methods. In both panels the data presented are the mean ± SEM for triplicate determinations obtained in one of at least three experiments performed for each mutant receptor. When not shown, SEM values fall within the symbols. Results are expressed as a percent of the maximal response (obtained at 10 mM Ca2+) for the WT hCaR. C, Intact cell ELISA was performed with cells transiently expressing WT hCaR, R680A, C677A, and E/D671,674A mutant receptors or untransfected control cells to measure cell surface expression levels using anti-hCaR 6D4 antibody as described in Materials and Methods. The OD 525 nm measured for the untransfected controls (0.127 ± 0.013) has been subtracted from the means of triplicate determinations. Results are expressed as a percent of the maximal WT hCaR OD value. The error bars are the SEM for these values. Two independent experiments gave similar results.

 
To assess whether substitution of both acidic residues may have any impact on [Ca2+]o-elicited response, we generated a double mutant E/D671,674A. Immunoblot analysis and Endo-H digestion showed this mutant receptor expressed both 150- and 130-kDa bands, and the presence of Endo-H-resistant 150-kDa band confirmed that this mutant receptor is expressed at the cell surface (Fig. 2Go). However, we observed some reduction in the intensity of the cell surface-expressed 150-kDa band, compared with the wild-type band. In functional PI assay (Fig. 3AGo), the double-mutant E/D671,674A displayed markedly attenuated [Ca2+]o-elicited response with a right-shifted dose-response curve (EC50 = 6.6 mM), with a maximal response of about half that found for the wild-type receptor (Table 1Go), which may be due to in part to poor cell surface expression. To further confirm that lack of or reduced functional response of C677A, R680A, and E/D671,674A were due to defects in cell surface expression of these mutants, their expression was assessed by quantitative intact-cell ELISA (Fig. 3CGo). Transient transfection with the wild-type hCaR yielded an ELISA signal that was 4- to 5-fold higher than the untransfected HEK293 cells. Surface expression of R680A was reduced approximately 30% and E/D671,674A was less than 50% of wild-type hCaR, whereas cell surface expression of C677A was negligible. These results confirm that C677A mutant receptor did not reach the cell surface and thus failed to respond to Ca2+, and R680A showed reduced cell surface expression, whereas the E/D671,674A mutant receptor expression at the cell surface was significantly reduced, compared with the wild-type hCaR, suggesting that lower cell surface expression of R680A and E/D671,674A may have contributed at least in part to reduce these mutant receptors activities. Taken together, these results suggest that single-point mutation of individual acidic or basic residues of Glu671, Asp674, Arg678, and Arg680 may not be crucial for the hCaR activity, whereas double mutations of Glu671 and Asp674 had greater impact on receptor activity.

Expression patterns and functional properties of EL2 mutant hCaRs
EL2 of the hCaR contain a single cysteine, a single basic residue, and five closely clustered acidic residues (Fig. 1Go). Single-point mutations of these residues were first created to understand the role of these residues in the context of the full-length hCaR. R752A and C765A replaced the only basic residue and a cysteine residue, respectively. E755A, E757A, D758A, E759A, and E767A each replaced a single residue of closely clustered acidic residues in EL2 by Ala. The expression patterns of these mutants were analyzed by the Endo-H digestion and immunoblotting experiments shown in Fig. 4Go. These results confirm that all mutants except C765A displayed a doublet pattern of immunoreactivity, and the band intensity of the Endo-H-resistant 150-kDa band of most of these mutants was similar to the wild-type hCaR. Similar to C677A, C765A receptor showed a single 130-kDa band and Endo-H digestion reduced the size of this expressed band completely, indicating that these were intracellular-trapped receptor forms. In PI assay, receptor bearing a basic residue mutation, R752A, exhibited a maximal response to [Ca2+]o that is similar to the wild type with an EC50 of 4.1 ± 0.05 mM (Fig. 5AGo and Table 2Go.). Like C677A, EL2 Cys mutant, C765A, also showed no significant response even at 10 mM [Ca2+]o concentration. All single acidic residue point mutants except E767A, i.e. E755A, E757A, D758A and E759A, exhibited quite similar sensitivity to [Ca2+]o as the wild-type receptor (Fig. 5BGo). In contrast, the E767A mutant receptor exhibited a substantial increase in its sensitivity to [Ca2+]o (EC50 of 2.1 ± 0.2 vs. wild-type EC50 4.2 ± 0.2 mM) with about 20% reduction in maximal response, compared with wild-type receptor (Table 2Go).



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  		FIG. 4. Cell surface expression of EL2 mutants of the hCaR. Eighteen micrograms of detergent-extracted membranes per lane for all other samples were analyzed by immunoblot of SDS-PAGE for WT hCaR and all the indicated EL2 mutant receptors expressing in HEK293 cells as described in Fig. 2Go. The positions of the 150- and 130-kDa forms and the Endo-H-digested nonglycosylated band migration (shown as an asterisk) are indicated on the right. The blot is representative of several similar experiments.

 


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  		FIG. 5. [Ca2+]o saturation of PI hydrolysis in HEK-293 cells expressing wild-type (WT) hCaR or EL2 mutant hCaR. A, HEK293 cells were transfected with cDNAs encoding WT ({blacktriangleup}), R752A ({diamond}), and C765A ({bullet}), and PI hydrolysis in response to the indicated concentrations of Ca2+ was performed as described in Fig. 3AGo. B, HEK293 cells were transfected for transient expression of WT hCaR ({blacksquare}), and the acidic residue mutants E755A ({diamond}), E757A ({bullet}), D758A ({blacktriangledown}), E759A ({circ}), E767A (), E/E755,757A ({blacktriangleup}), D/E758,759A ({triangledown}), E3A ({square}), and E/D5A ({triangleup}) and PI hydrolysis in response to the indicated concentrations of Ca2+ was measured. In both panels the data presented are the mean ± SEM for triplicate determinations obtained in one of at least three experiments performed for each mutant receptor. When not shown, SEM values fall within the symbols. Results are expressed as a percent of the maximal response (obtained at 10 mM Ca2+) for the WT hCaR. C, Intact cell ELISA was performed with cells transiently expressing WT hCaR, C765A, E767A, E3A, and E/D5A mutant receptors or untransfected control cells to measure cell surface expression levels as described in Fig. 3CGo. The value obtained for untransfected control cells of 0.156 ± 0.006 OD 525 nm has been subtracted from all other values, and results are expressed as a percent of maximal WT hCaR OD value. Data shown are the means and SEM of triplicate determinations from a single experiment. Two independent experiments gave similar results.

 
Because several EL acidic residues may contribute to Ca2+-binding in 7TM and also because our previous work had shown mutation of all these five acidic residues in EL2 in the context of the amino-terminal ECD-deleted construct T903Rhoc significantly attenuated Ca2+ and other cationic response in presence of NPS-R568 (9), we examined the [Ca2+]o-elicited functional responses of different combinations of multisite acidic mutants including two double-site mutants, E/E755,757A and D/E758,759A, and two additional multiple-site mutants, E3A and E/D5A, in the context of the full-length hCaR. E3A and E/D5A substituted alanines for Glu755, Glu757, and Glu767 or replaced all five EL2 acidic residues with alanines, respectively. All multisite mutants, except E/D5A, displayed doublet band patterns in immunoblotting experiments, and the expression levels of the Endo-H-resistant 150-kDa band of these mutants except E/D5A showed comparable band intensity as the wild-type hCaR (Fig. 4Go). Of these multisite mutants, the two double mutants, E/E755,757A and D/E758,759A, exhibited only marginally different sensitivity to [Ca2+]o, compared with the wild-type hCaR in functional PI assay (Fig. 5BGo and Table 2Go.). The E/E755,757A mutant receptor showed slightly increased sensitivity to [Ca2+]o, whereas the D/E758,759A mutant exhibited slightly reduced affinity for [Ca2+]o with neither mutations altering maximal response from that of wild-type hCaR (Table 2Go.). E3A mutant substituting alanines for Glu755, Glu757, and Glu767 showed similar activating effect as E767A and did not show further increase in [Ca2+]o sensitivity over E767A activation profile with an EC50 value of 2.3 mM.

In contrast, the signaling property of E/D5A that eliminated all five acidic residues was dramatically altered. The maximal PI hydrolysis response was diminished to 35% of the wild-type response, and the shape of the Ca2+-saturation curve changed to hyperbolic from the steeply sigmoidal saturation profile of wild-type hCaR. Because C765A, E767A, E3A, and E/D5A had greatest impact in Ca2+-elicited functional assays, to determine whether these functional responses are related to differences in levels of cell surface expression, we quantified the impact of the C765A, E767A, E3A, and E/D5A mutations on cell surface expression by intact-cell ELISA (Fig. 5CGo). The data show that, compared with the wild-type hCaR, E767A and E3A showed approximately 33–38% and 15–20% reduction, respectively, in cell surface expression. Cell surface expression of E/D5A was 75% reduced, and cell surface expression of C765A was not detectable. These results confirmed that E767A and E3A activating effects were not due to higher cell surface expression levels, compared with the wild-type receptor. Moreover, in both Endo-H digestion and immunoblot analysis and in intact cell ELISA (Figs. 4Go and 5CGo), we observed highly diminished cell surface expression of E/D5A. Interestingly, even with significantly lower cell surface expression, HEK 293 cells expressing E/D5A mutant receptor showed 2- to 3-fold higher PI hydrolysis at basal Ca2+ (0.5 mM), compared with the wild-type hCaR expressing cells (Fig. 5BGo). In contrast, an identical mutant recently reported by Hu et al. (10) with all five acidic residues in EL2 (5A mutant) completely abolished [Ca2+]o response and did not express at the cell surface. We believe differences in transfection efficiency and conditions of the HEK-293 cells in culture may have contributed to these differences in experimental observations.

Expression patterns and functional properties of EL3 mutant hCaRs
A single positively charged residue Lys831 and a negatively charged residue Glu837 in EL3 were changed to alanine individually to create K831A and E837A mutant receptors, and expression patterns along with the functional responses of these mutants were analyzed. Immunoblotting experiment using 6D4 antibody showed that both K831A and E837A mutants expressed both the 150- and 130-kDa bands similar to the wild-type hCaR (Fig. 6Go). Also, Endo-H digestion minimally reduced the size of the 150-kDa band of both wild-type hCaR and mutant receptors and reducing the size of the 130-kDa band completely. The intensity of the cell surface-expressed Endo-H-resistant 150-kDa band of K831A was comparable with the wild-type hCaR, whereas that for E837A was somewhat reduced (Fig. 6Go). [Ca2+]o-induced PI hydrolysis showed the [Ca2+]o-induced PI dose-response curve was left shifted for K831A with an EC50 value of approximately 2.8 mM, compared with 4.2 mM for the wild-type hCaR (Fig. 7AGo), whereas E837A response was similar to the wild-type hCaR with an EC50 of 4.0 mM but with a 26% reduction in maximal response (Fig. 7Go. B). Cell surface ELISA confirmed that K831A expressed at similar levels at the cell surface and E837A cell surface expression was lower, compared with the wild-type hCaR (Fig. 7CGo). These results confirm that gain-of-function activity of K831A is not due to significantly higher cell surface expression of this mutant receptor and that the decrease in maximal response of E837A in PI assay may in part be due to lower cell surface expression of this mutant receptor.



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  		FIG. 6. Cell surface expression of EL3 mutant receptors of the hCaR. Cell surface expression was analyzed for 18 µg of crude membrane extract for K831A and E837A EL3 mutant hCaR constructs as described in Fig. 2Go. The positions of the 150- and 130-kDa forms and the Endo-H-digested nonglycosylated band migration (asterisk) are shown on the right. This blot is representative of two independent experiments that gave similar results.

 


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  		FIG. 7. [Ca2+]o saturation of PI hydrolysis in HEK-293 cells expressing wild-type (WT) hCaR or EL3 mutants K831A and E837A. A, HEK293 cells were transfected for transient expression of WT hCaR ({blacktriangleup}) and the basic residue mutant K831A ({diamond}), and PI hydrolysis in response to the indicated concentrations of Ca2+ was measured as described in Fig. 3AGo. B, HEK293 cells were transfected for transient expression of WT hCaR ({blacktriangleup}) and the acidic residue mutants E837A ({square}), and PI hydrolysis in response to the indicated concentrations of Ca2+ was measured as described in Fig. 3AGo. In both panels the data presented are the mean ± SEM for triplicate determinations obtained in one of at least three experiments performed for each mutant receptor. When not shown, SEM values fall within the symbols. Results are expressed as a percent of the maximal response (obtained at 10 mM Ca2+) for the WT hCaR. C, Intact cell ELISA was measured with 6D4 antibody for HEK-293 cells expressing WT hCaR, K831A, E837A, or untransfected HEK-293 cell. Background OD 525 nm value of 0.134 ± 0.004 has been subtracted from all values and expressed as a percent of maximal WT hCaR OD values. Results shown are the means of triplicate determinations. Two independent experiments gave similar results.

 
Analysis of signal transduction by mutant receptors replacing Glu767 and Lys831with different amino acids
Because Glu767 and Lys831 were the two residues whose alanine mutations produced activating affects, we wished to determine the role of the side chains of Glu767 and Lys831. We substituted Glu767 with Asp (E767D), Gln (E767Q), or Lys (E767K) and replaced Lys831 with Glu (K831E) or Gln (K831Q). Expression analysis by immunoblotting with anti-hCaR 6D4 antibody showed these mutants expressed both the cell surface-expressed 150-kDa forms and intracellularly trapped 130-kDa forms (Fig. 8Go, A and B). As shown in Fig. 9AGo, the responses of E767Q and E767K mutant receptors were similarly activating as the E767A mutant with left-shifted dose-response curves and EC50 values of 2.0 and 2.1 mM, respectively (Table 2Go). Substitution with a like-charged residue in the E767D mutant yielded somewhat right-shifted dose-response curve with EC50 value of 5.1 mM. Similarly, replacement of Lys831 with Glu (K831E) and Gln (K831Q) generated [Ca2+]o-induced left-shifted receptor responses with EC50 values of 3.0 and 3.8, respectively. The K831Q dose-response curve was not as much left-shifted as K831A and K831E and also showed a consistently higher maximal response, compared with the wild-type hCaR (Table 3Go).



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  		FIG. 8. Cell surface expression of Asp767 and Lys831 mutant hCaR receptors. A, Cell surface expression was analyzed for 18 µg crude membrane extract for HEK293 cells transiently expressing wild-type (WT) hCaR, E767A, E767D, E767K, and E767Q as described in Fig. 2Go. B, Cell surface expression was analyzed for 18 µg crude membrane extract for HEK293 cells transiently expressing WT hCaR, K831A, K831E, K831Q, and E767K/K831E as described in Fig. 2Go. The migration of the 150- and 130-kDa bands of the hCaR are indicated.

 


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  		FIG. 9. [Ca2+]o saturation of PI hydrolysis in HEK-293 cells expressing wild-type (WT) hCaR or E767 and K831 mutations. A, HEK293 cells were transfected with cDNAs encoding WT ({blacktriangleup}), E767A ({triangleup}), E767D ({triangledown}), E767K ({bullet}), and E767Q ({blacksquare}) mutant hCaR constructs, and PI hydrolysis in response to the indicated concentrations of Ca2+ was measured as described in Fig. 3Go. B, HEK293 cells were transfected with cDNAs encoding WT ({blacktriangleup}), K831A ({square}), K831E ({bullet}), K831Q ({triangledown}), and E767K/K831E ({diamond}) mutant hCaR constructs, and PI hydrolysis in response to the indicated concentrations of Ca2+ was measured as described in Fig. 3Go A. In both panels the data presented are the mean ± SEM for triplicate determinations obtained in one of at least three experiments performed for each mutant receptor. When not shown, SEM values fall within the symbols. Results are expressed as a percent of the maximal response (obtained at 10 mM Ca2+) for the WT hCaR. C and D, Intact cell ELISA was performed with cells transiently expressing WT hCaR and different replacement mutants of Glu767 and Lys831 with different amino acids from the same transient transfection experiments shown in the immunoblot in Fig. 8Go. Untransfected cells were used to measure background signal. The value obtained for untransfected control cells of 0.452 ± 0.006 OD and 0.152 ± 0.005, respectively, at 525 nm has been subtracted from all other values from individual experiments in C and D, and results are expressed as a percent of maximal WT hCaR OD value. Data shown are the means and SEM of triplicate determinations from a single experiment. Two independent experiments gave similar results.

 
Because both E767K and K831E were found to be activating and seemed to reside on the same side that may connect EL2 and EL3, to determine whether the side chains of Glu767 and Lys831 forms a salt-bridge, we tested a double-mutant E767K/K831E, with the Glu767 and Lys831 residues switched. If they form a salt-bridge, the E767K/K831E mutation would be predicted to restore wild-type phenotype. Instead, unlike other Glu767 and Lys831 mutants, the E767K/K831E double mutant expressed a very faint 150-kDa band and mostly the 130-kDa band (Fig. 8BGo). Similar to C677A and C765A mutants, this single 130-kDa band was sensitive to Endo-H digestion, and Endo-H digestion reduced the size of this expressed band completely (data not shown). Consistent with immunoblot data, this mutant also showed no detectable [Ca2+]o-induced PI hydrolysis even at 10 mM [Ca2+]o concentration (Fig. 9BGo). Whole cell surface ELISA experiments (Fig. 9Go, C and D) were performed to confirm that the activating effects shown by different E767 and K831 mutants in functional PI assays were not due to significantly higher cell surface expression, compared with the wild-type receptor. The data showed that a small fraction (15%) of E767K/K831E reached the cell surface, and the levels of cell surface expression of the remaining activating mutants were either similar or lower than the wild-type receptor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The precise mechanism of activation for any family 3 GPCR member remains largely unknown. Whereas the large amino-terminal ECD structures certainly contain a primary binding site(s) for activating ligands, the mechanism of transmission of ligand recognition to G protein activation by the intracellular structures of the 7TM domain has not been elucidated. Because disulfide linkages and ionic interactions within ELs may play a role in hCaR activation and our previous study also indicated the presence of additional Ca2+ recognition site(s) within 7TM core of the hCaR (9), in this paper we investigated the roles of the basic and acidic residues along with two highly conserved cysteines in the EL structures in the hCaR activation. In comparison with many rhodposin-like GPCRs, the length and amino acids of the ELs of the hCaR is highly variable. When we compared ELs of several GPCRs, we observed that several GPCRs contain two small EL1 and EL2 and the third large EL3; in contrast, the EL2 of the hCaR is the largest with no apparent conservation in the length of these loops relative to its position, compared with rhodopsin. Despite overall differences in EL lengths, two highly conserved Cys residues in EL1 and EL2 that are known to be linked by disulfide bond in rhodopsin and several other GPCRs seems to be present in similar locations in the hCaR. In the present study, we found that mutation of Cys677 and Cys765 in EL1 and EL2 of the hCaR, respectively, cause lack of cell surface expression (cell surface ELISA) and abnormal processing (Endo-H sensitivity/immunoblotting) of the mutant receptors.

These results suggest both these cysteines are critical in maintaining the overall conformation of the hCaR but do not allow us to distinguish whether mutation of each of these cysteines disrupts a disulfide bridge with one of the several Cys residues present in the ECD or with each other. We note that in some GPCRs these two corresponding Cys residues have been suggested to pair with another Cys rather than with each other (14, 15). In either case, we speculate that disruption of such a disulfide linkage would lead to misfolding and processing defects of Cys mutants similar to those reported for other Cys mutations in the hCaR ECD (20). Interestingly, in addition to Cys mutations, multiple mutations (E/D671,674A, E/D5A, and E767K/K831E) in the EL1, EL2, and EL3 also lead to significantly reduced cell surface expression of these mutant receptors structures, again indicating misfolding and processing defects of these mutant receptors. Taken together, these results suggest that several structural elements in EL1, EL2, and EL3 combine to maintain the overall conformation of the 7TM structure including one or more disulfide bridges and possibly ionic bonds between some charged residues, which are in turn important for receptor folding and processing.

Of all of the charged amino acid residues in the ELs, only Glu767 and Lys831 when individually mutated lead to significant alteration of the signaling properties of the hCaR. Our data show both E767A and K831A are activating or gain-of-function mutations. Mutations of Glu767 (E767A, E767Q, or E767K) are strongly activating, whereas mutations of Lys831 (K831A, K831E, or K831Q) were less strongly activating. The EC50 values of these gain-of-function mutations at Glu767 position ranged between 1.8 and 2.4 mM, whereas, activating mutations of Lys831 produced receptors with EC50 values of 2.8 and 3.0 mM. Replacement of these positions with various amino acids suggests that both Glu767 and Lys831 participate in ionic contacts. Because Asp in place of Glu767 position displayed nearly wild-type activity and Gln, Lys, or Ala substitution at this position was strongly activating, it is likely that the carboxylate group of Glu767 is involved in an ionic interaction rather than hydrogen bonding. Likewise, changing Lys831 to Ala or Glu was similarly activating, whereas to Gln generated a mutant receptor response similar to wild-type hCaR. Hence, Lys831 also appears to participate in an ionic interaction like Glu767 and both of which seems to be crucial for receptor activation. Whereas it was tempting to speculate that the Glu767 and Lys831 participated in an ionic bridge between EL2 and EL3, the E767K/K831E double mutant refuted this. We expected either this mutation would restore an ionic bridge between these two positions and generate a wild-type phenotype or the combination of two gain-of-function mutations might further increase the sensitivity to [Ca2+]o as seen for several activating mutations of the hCaR (21). Surprisingly, this mutant receptor failed to reach the cell surface and remained intracellularly trapped as Endo-H sensitive, improperly glycosylated forms. Thus, we argue that no direct ionic contact exists between Glu767 and Lys831 because in such case switching these charged amino acid positions might not have misfolded the E767K/K831E mutant receptor structure. Instead, we suspect that repulsive interactions of these two amino acids with similar charged residues at respective switched positions may have contributed to the misfolding/processing defect of the mutant protein. This raises the possibility that two distinct ionic interactions between Glu767 with a positive charged residue and Lys831 with a negative charged residue, respectively, may be critical to maintain an inhibited conformation of the 7TM domain of the hCaR, and disruption of not one but both is necessary to misfold the E767K/K831E mutant receptor structure.

In this context, it is important to mention that our result is similar to that recently reported by Hu et al. (10) for E767A (activating mutation) and some other alanine substitution mutants of acidic residues in ELs but differ importantly for two other acidic residue mutations in EL2. Unlike our results, this study reported that mutation of two other residues, Asp758 and Glu759, individually to alanine or in combination in the 4A mutant (Glu755, Glu757, Asp758, and Glu759) showed strong activation of PI hydrolysis with corresponding EC50 values less than 1 mM. In contrast, we found no evidence that individual mutation of Asp758 or Glu759 produced activated receptors; these results are not consistent with the previous study (10), but a reason for this difference remains to be established. We found that the individual D758A and E759A mutants as well as the double-mutant D/E758,759A exhibited nearly wild-type phenotype with EC50 values close to that of the wild-type hCaR. These data strongly suggest that alanine substitutions at these residues cause no significant gain-of-function response by these receptor mutants, compared with the wild-type and thus they are not activating mutations. Based on these results, we argue that instead of three acidic residues in EL2 as reported (10), only Glu767 is critical to constrain the 7TM domain in a negative conformation. However, multiple mutations of acidic residues in EL2 in the E/D5A mutant profoundly alter the cell surface expression and signaling phenotype of this mutant receptor, abolishing all observable cooperativity in activation and producing the lowest EC50 value for Ca2+-stimulated PI hydrolysis of all constructs tested in this report.

A variety of amino acid substitutions at different positions of GPCRs can lead to their constitutive activation (22); the inactive, unliganded form of the receptors would be subject to structural constraints, which could be released by a number of different mutations. To date, 25 missense activating mutations of the hCaR and one large in-frame deletion of the C-terminal domain have been reported in patients with autosomal dominant hypocalcemia and/or sporadic hypocalcemia shown to cause increased sensitivity to [Ca2+]o rather than constitutive activity (21). Fifteen of these mutations are localized within the ECD and at least six in the transmembrane helices. Within the ECD, several of these activating mutations are present within the Ala116-Pro136 region, which is believed to be the disulfide-linked dimer interface, and play important role in maintaining the inactive conformation of the hCaR ECD (23, 24). In contrast, only three naturally occurring activating mutations, Q681H, F832S, and A835T, have been identified in the EL1 and EL3, and A835T activating mutation in EL3 showed very similar activating effect (EC50 of 2.86) as the K831A mutation reported in this paper (21, 25). Thus, the E767A and K831A mutations along with other amino acid substitutions that cause gain-of-function effects define these two new positions in EL2 and EL3, respectively, as potential residues for such activating mutations in human endocrine diseases.

Analysis of the functional characteristics of large ECD-containing glycoprotein hormone receptor family (family 2), particularly TSH and LH receptors, lead to the hypothesis that the ECD domain of these and other family 2 GPCR structures could act as an inhibitory constraint on the 7TM domains (26, 27). However, unlike the TSH receptor, there is little evidence that the unliganded ECD plays such a role for the hCaR. A truncation mutant lacking most of the hCaR ECD (T903-Rhoc) with an intact 7TM domain seems to show no significant constitutive activity (9, 10, 28). With these limited data, it is difficult to confirm whether the ECD of the hCaR can act as an inhibitory constraint; however, an allosteric interaction between the structural elements in the ECD and 7TM helices via ELs for the activation of the hCaR still remains a viable possibility. Thus, we speculate that Glu767 and Lys831 may be linked to ECD by ionic bonds, and a mutation that disrupt one of these contacts may contribute to improper allosteric interaction of the ECD with the 7TM domain and thus lead to gain-of-function activity. A reduction of cooperative responsiveness as judged by Hill coefficient numbers of some of the Glu767 mutations with strong activating affects (Table 2Go) may be indicative of such improper allosteric interactions between the ECD and 7TM domain. However, in the absence of any Ca2+-binding assay, we were unable to determine whether these gain-of-function activities of the mutant receptors are due to higher affinity for ligand binding or due to conformational changes independent of changes in ligand binding affinities, but the results are compatible with a receptor activation model in which allosteric interactions between the ligand-bound ECD and the ELs of the 7TM domain could lead to activation of G protein signaling.


   Footnotes
 
Abbreviations: [Ca2+]o, Extracellular calcium; ECD, extracellular amino-terminal domain; EL, extracellular loop; Endo-H, endoglycosidase-H; FBS, fetal bovine serum; GPCR, G-protein-coupled receptor; hCaR, human extracellular Ca2+ receptor; HEK, human embryonic kidney; mGluR, metabotropic glutamate receptor; NPS-R568, (R)-N- (3-methoxy-{alpha}-phenylethyl)-3-(2'-chlorophenyl)-1-propylamine hydrochloride; PI, phosphoinositides; 7TM, seven transmembranes.

Received December 4, 2003.

Accepted for publication April 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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