The Extracellular Calcium-sensing Receptor Dimerizes through Multiple Types of Intermolecular Interactions*

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca2+] o )-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys129 and Cys131 in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys129 and Cys131, showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.

The extracellular calcium ([Ca 2ϩ ] o )-sensing receptor (CaR) 1 is a G protein-coupled receptor that plays a key role in mineral ion homeostasis by sensing small perturbations in the level of [Ca 2ϩ ] o and modulating the functions of parathyroid and kidney so as to restore [Ca 2ϩ ] o to its normal level (1). Like some G protein-coupled receptors (2,3), recent studies have shown that the CaR on the surface of receptor-transfected cells forms disulfide-linked dimers (4) through cysteines within its extracellular domain (5,6). However, one study suggested that two cysteines at positions 101 and 236 mediate intermolecular disulfide linkages (5), whereas another showed that two cysteines at positions 129 and 131 are involved in disulfide-linked dimerization (6). The resultant mutant receptors lacking either of these two pairs of cysteine residues were suggested to form monomers rather than dimers in transfected cells (5,6). These putatively monomeric CaRs had no biological activity in one case (5) or exhibited increased sensitivity to [Ca 2ϩ ] o in the other (6). Thus there is currently no consensus as to the key cysteine residues within the CaR extracellular domain that mediate dimerization.
Our earlier studies indicated that the CaR may dimerize through noncovalent interactions between transmembrane domains in addition to doing so via covalent disulfide bonds within its extracellular domain (5,6), as suggested by our studies on naturally occurring inactivating mutations causing familial hypocalciuric hypercalcemia (7,8). We showed that under reducing conditions, most mutant receptors, including those with almost undetectable mature forms such as R66C and R680C, show substantial amounts of SDS-resistant dimeric forms in addition to monomeric forms in SDS-containing polyacrylamide gels. In contrast, P747frameshift, a single-base deletion in codon 747 resulting in a truncated receptor lacking the second extracellular loop and the remainder of the C terminus, including the fifth, sixth, and seventh transmembrane domains, shows only a single monomeric species and no detectable higher molecular weight forms. Interestingly, a consensus dimerization motif (9) for noncovalent hydrophobic interactions is present in the CaR within TM5, that is missing in the P747frameshift.
In the present studies, we have examined the roles of Cys 101 , Cys 129 , Cys 131 , and Cys 236 in mediating disulfide-linked dimerization of the CaR and of covalent dimerization in the functional interactions between CaR monomers within the dimeric CaR. Mutating both Cys 129 and Cys 131 slightly reduces cell surface expression of the CaR and abolishes most of the sulfhydryl-sensitive CaR dimerization on the cell surface. In contrast, mutating Cys 101 and Cys 236 severely reduces surface and overall expression of the receptor with no substantial conversion of dimers to monomers detected under nonreducing conditions. Furthermore, we show that the CaR still forms functional dimers even in the absence of these intermolecular disulfide linkages.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-Site-directed mutagenesis was performed using the approach described by Kunkel (10) to produce mutated receptors in which one or two cysteine residues present in the extracellular domain of the human CaR were mutated to serines. The dut-1 ung-1 strain of Escherichia coli, CJ236, was transformed separately with mutagenesis cassettes, as described previously (7). Uracilcontaining, single-stranded DNA was produced by infecting the cells with the helper phage, VCSM13 (Stratagene, La Jolla, CA). The singlestranded DNA was then annealed to a mutagenesis primer that contained the desired nucleotide change encoding a single point mutation flanked on both sides by wild type sequences. The primer was subsequently extended around the entire single-stranded DNA and ligated to generate closed circular heteroduplex DNA. DH5␣-or DH10B-competent cells were transformed with these DNA heteroduplexes, and incor-poration of the desired mutations was confirmed by sequencing the mutated cassettes. The resultant mutated cassettes were cloned into the FLAG-tagged or nontagged receptor in pcDNA3 (Invitrogen), as described previously (7).
Construction of Mutant CaRs with Double Mutations of C101S/ C236S-Cassette 1, carrying C101S, was doubly digested with BspEI and NheI, and the larger fragment obtained from the above digestions was ligated to the smaller fragment resulting from digestion of the CaR carrying C236S with BspEI and NheI.
Construction of C129S/C131S-containing CaRs with Inactivating Mutations-Cassette 1 carrying C129S/C131S was doubly digested with KpnI and BspEI, and the smaller fragment obtained from the above digestion was ligated to the larger fragment resulting from digestion of the CaR carrying E297K or A877Stop with KpnI and BspEI.
Transient Expression of CaRs in HEK293 Cells-The DNA for transfection was prepared using the Midi Plasmid Kit (Qiagen). Lipo-fectAMINE (Life Technologies, Inc.) was employed as a DNA carrier for transfection (11). The human embryonic kidney (HEK293) cells used for transient transfection were provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT) and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal bovine serum (Hyclone). The DNA-liposome complex was prepared by mixing DNA and Lipo-fectAMINE in OPTI-MEM I reduced serum medium (Life Technologies, Inc.) and incubating the mixture at room temperature for 30 min. The DNA-lipofectAMINE mixture was then diluted with OPTI-MEM I reduced serum medium and added to 90% confluent HEK293 cells plated on 13.5 ϫ 20.1-mm glass coverslips using 2.5 g of DNA. After a 5 h incubation at 37°C, equivalent amounts of OPTI-MEM I reduced serum medium with 20% fetal bovine serum were added to the medium overlying the transfected cells, and the latter was replaced with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 24 h after transfection. The expressed [Ca 2ϩ ] o -sensing receptor protein was assayed 48 h after the start of transfection. To perform coexpression of two receptors, 1.25 g of each of the two cDNAs were mixed and used to transfect HEK293 cells.
Biotinylation of Cell Surface Forms of the CaR and Cross-linking of Multimeric Receptors-Before preparing whole cell lysates, intact HEK293 cells transiently transfected with FLAG-tagged CaR were washed twice with phosphate-buffered saline and treated with 1 mM ImmunoPure Sulfo-NHS-Biotin (Pierce), a membrane-impermeant biotinylation reagent, at room temperature with constant agitation for 30 min to biotinylate the proteins on the cell surface. The reaction was then quenched by incubating the cells in 1 M Tris-HCl, pH 7.5, for 5 min. For cross-linking experiments, we added an appropriate amount of bis(sulfosuccinimidyl) suberate (BS 3 ), a noncleavable, membrane-impermeant cross-linker, into the labeling solution with ImmunoPure Sulfo-NHS-Biotin.
Immunoprecipitation of FLAG-tagged CaRs-To a microcentrifuge tube, 5 g of anti-FLAG M2 monoclonal antibody, 400 l of H 2 O, 500 l of 2ϫ immunoprecipitation buffer (see above), and 100 l of total lysate containing ϳ250 or 500 g of total proteins were added. The mixture was incubated at 4°C for 1 h. To the mixture was then added 5 l of an anti-mouse IgG (Sigma). The incubation was continued for an additional 30 min at 4°C. To the mixture was subsequently added 50 l of 10% protein A agarose (Life Technologies, Inc.) for a further 30-min incubation at 4°C. The protein A-agarose was washed three times with 1ϫ immunoprecipitation buffer, and the immunoreactive species were subsequently eluted in 45 or 60 l of 2ϫ electrophoresis sample buffer at 65°C for 30 min. In a given experiment, the same amount of total protein for each sample was used for immunoprecipitation and was eluted with the same volume of SDS-sample buffer. The receptor of interest was detected by Western analysis.
Western Analysis of the Human CaR Expressed in Whole Cells and on the Cell Surface-If not specified, 15 l of eluted, immunoprecipitated sample was subjected to SDS-containing polyacrylamide gel electrophoresis (PAGE) (12) using a linear gradient of polyacrylamide (3-10%). The proteins on the gel were subsequently electrotransferred to a nitrocellulose membrane. After blocking with 5% milk, the forms of the receptor present on the cell surface were detected using an avidinhorseradish peroxidase conjugate (Bio-Rad) followed by visualization of the biotinylated bands with an enhanced chemiluminescence (ECL) system (PerkinElmer Life Sciences). After removal of the avidin using the recommended procedure for stripping the blots (Amersham Pharmacia Biotech), all forms of the CaR on the same blot were detected using an anti-CaR antiserum (4641 or 4637, polyclonal antisera raised against two peptides within the extracellular domain of the CaR, kindly provided by Drs. Forrest Fuller and Rachel Simin at NPS Pharmaceuticals, Salt Lake City, UT) followed by a secondary, horseradish peroxidase-conjugated goat anti-rabbit antibody and then an ECL system (Amersham Pharmacia Biotech).

Measurement of [Ca 2ϩ ] i by Fluorimetry in Cell
Populations-Coverslips with HEK293 cells previously transfected with the appropriate CaR cDNAs were loaded for 2 h at room temperature with fura-2/AM in 20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , 0.1% bovine serum albumin, and 0.1% dextrose and then washed once with a bath solution (20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% dextrose, and 0.1% bovine serum albumin) at 37°C for 20 min. The coverslips were then placed diagonally in a thermostatted quartz cuvette containing the bath solution, using a modification of the technique employed previously in this laboratory (13). Extracellular calcium was increased stepwise to give the desired final concentrations with additions of [Ca 2ϩ ] o in increments of 1 mM, which were followed by 5 mM increments after achieving a level of 5.5 mM [Ca 2ϩ ] o and 10 mM increments after reaching a level of 20 mM [Ca 2ϩ ] o . Excitation monochrometers were centered at 340 and 380 nm, and emission light was collected at 510 Ϯ 40 nm through a wide-band emission filter. The 340/380 excitation ratio of emitted light was used to calculate [Ca 2ϩ ] i as described previously (13).
Statistics-The mean EC 50 values for the various wild type or mutant receptors determined in response to increasing concentrations of [Ca 2ϩ ] o were calculated from the EC 50 values for all of the individual experiments and were expressed with the S.E. as the index of dispersion. Comparison of the EC 50 values was performed using analysis of variance or Duncan's multiple comparison test (14) (p Յ 0.05). Each of the experiments described above in the experimental protocols was generally performed at least three times.   (15), one group of mutant receptors carrying C129S, C131S, C437S, C449S, or C482S (Group I mutations in Fig. 1), exhibit nearly normal levels of expression on the cell surface and in whole cell lysates (Fig. 1, lanes 2, 3, and 5-7). Of these receptors, C437S and C482S (Fig. 1A, lanes 5 and 7) have slightly higher cell surface expression than wt (Fig. 1A, lane 1), whereas C129S, C131S, and C449S (Fig. 1A, lanes 2, 3, and 6) have somewhat lower cell surface expression than wt.

Characterization of CaRs Carrying Single Cys to Ser Mutations in the Extracellular
In contrast, another group of mutant receptors carrying C60S, C101S, C236S, or C598S (Group II mutations in Fig. 2), have marked reductions in their surface expression ( Fig. 2A). In Fig. 2, lanes 3-6, twice as much of the samples were loaded as those in lanes 1 and 2. The low apparent immunoreactivity of C236S toward the anti-CaR antibody, 4641, in Fig. 2B, lane 5, is likely due to the fact that Cys 236 is the last amino acid in the peptide (Ala 214 -Cys 236 ) against which 4641 was raised and may be important for the binding of 4641 to the receptor. As shown below, C236S has a similar level of overall expression to that of C598S when it was detected with another anti-CaR antibody, 4637, which was raised against a peptide consisting of amino acids 344 -358 in the extracellular domain of the CaR. Whereas C598S (Fig. 2B, lane 2) had a much lower mature form than wt, two other mutant receptors, C60S and C101S, were even lower (Fig. 2B, lanes 3 and 4). Note that twice the amount of eluted, immunoprecipitated samples were loaded in Fig. 2, lanes 3 and 4, as in Fig. 2, lanes 1 and 2. The mature forms of these mutant receptors are N-glycosylated with complex carbohydrates, corresponding to the upper bands of the doublets between 127 and 200 kDa (7).
Under nonreducing conditions, all of the mutant receptors with a single point mutation, behaving like wt, were present mostly as dimers or higher multimeric forms on SDS-PAGE ( Fig. 3 and 4) when we detect either only the surface forms or all forms of the receptor. However, longer exposure reveals trace amounts of monomeric CaRs for some of the mutant receptors (e.g. C129S, C131S, and C482) on blots stained for cell surface receptor proteins (Fig. 3A, lanes 2, 3, and 6). In contrast to previous studies with C60S, C101S, and C236S (5,15), no significant amounts of the monomeric species of these three mutant receptors were detected either on the cell surface or in the whole cell lysates (Fig. 4, lanes 2-4). As shown in Fig.  4B, lanes 4 and 5, C236S, has a similar level of overall expression to that of C598S when it was detected with 4637. Thus dimerization of wt appears to involve more than one intermolecular disulfide bond, necessitating mutation of two or more cysteines to identify those forming intermolecular disulfide bonds.
Construction and Characterization of Mutant CaRs with More than One Cys to Ser Mutation in the Extracellular Domain-To identify cysteines forming intermolecular disulfide bonds, we constructed mutant receptors with two or more mutations of cysteines to serines but focused on those that had no detrimental effects on either receptor expression or function when studied as single mutations. We found that the mutant receptor, C129S/C131S, had a substantial level of expression on the cell surface (Fig. 1A, lane 4), although it was slightly lower than those of the mutant receptors with C129S or C131S alone (Fig. 1A, lanes 2 and 3). The activity of the mutant receptor, C129S/C131S, was also affected more than those of the two singly mutated receptors, C129S and C131S (Table I). C129S/C131S became even more sensitive to [Ca 2ϩ ] o , with a lower EC 50 than that of C129S and similar to that of C131S. The cumulative maximal response of C129S/C131S was significantly lower than those of the singly mutated receptors.
Under nonreducing conditions, a substantial amount of monomeric C129S/C131S was detected when assessed by SDS-PAGE (Fig. 5, lane 2). Detection of the cell surface form of this receptor (Fig. 5A, lane 2) revealed that the monomeric receptor is the major species, unlike wt (Fig. 5A, lane 1). In contrast, reprobing of the same blot with an anti-receptor antibody, detecting both cell surface and intracellular forms of the receptor (Fig. 5B, lane 2), showed that the monomeric form of the receptor is a minor species. Thus, whereas a large fraction of the mutant receptor, C129S/C131S, no longer forms intermolecular disulfide bonds on the cell surface, the majority of intracellular receptor species either still forms intermolecular disulfide bonds or is resistant to dissociation of dimers to monomers during SDS-PAGE despite the lack of intermolecular disulfide linkages.
When we examined mutant receptors carrying triple, quadruple, or quintuple mutations, we found that, like C129S/ C131S (Fig. 5A, lane 2), substantial amounts of receptors containing both C129S and C131S no longer form intermolecular disulfide-linked dimers on the cell surface (Fig. 5A, lanes 3 and  4). However, the major species in the whole cell lysates are still multimers (Fig. 5B, lanes 3 and 4). Other receptors with multiple Cys to Ser mutations, containing either Cys 129 or Cys 131 , still form exclusively disulfide-linked dimers on the cell surface and in whole cell lysates (data not shown).
We also examined the properties of the mutant receptor with the two mutations, C101S and C236S (Figs. 2 and 4, lane 6). In contrast to a previously published study (5), the overall expression level of C101S/C236S (Fig. 4B, lane 6) is equivalent to that of C101S (Fig. 4B, lane 3) and much less than that of C236S (Fig. 4B, lane 4). In addition, we found the surface expression level of C101S/C236S (Fig. 2A, lane 6), although detectable, much less than the mutant receptor carrying either mutation alone ( Fig. 2A, lanes 4 and 5). Under nonreducing conditions, there are no detectable mature monomeric species of the CaR in whole cell lysates even though we detected a trace amount of its immature form when the blot for Fig. 4B was exposed for a much longer period of time (data not shown). This mutant receptor does show slight activity (Table I).
Thus Cys 129 and Cys 131 appear essential for formation of intermolecular disulfide bonds in most of the cell surface forms of the CaR, similar to the result of Ray et al. (6). However, we still detect a substantial amount of dimeric C129S/C131S on the cell surface (Fig. 5A, lane 2), even though it is a minor species relative to its monomeric counterpart. Next we examined whether the mutant receptor, C129S/C131S, which appears as a monomeric receptor in SDS-containing buffer without any dithiothreitol, may exist on the cell surface as noncovalently linked dimers. We utilized cross-linking of cell surface receptors and coimmunoprecipitation of cotransfected receptors as described previously (4).

Demonstration of Dimer Formation of the CaR on the Cell Surface in the Absence of Intermolecular Disufide Bonds-To
determine whether the mutant receptor, C129S/C131S, forms cell surface dimers, we stabilized any preexisting dimeric or higher multimeric forms of the receptor by covalently linking them with BS 3 , a noncleavable, membrane impermeant crosslinker, and surface-labeled the cells with Sulfo-NHS-Biotin. The surface-biotinylated and cross-linked cells were then lysed in the presence of iodoacetamide. The FLAG-tagged C129S/ C131S and wt were then immunoprecipitated, and the immunopurified CaRs were eluted with DTT-containing SDS sample buffer. Increasing the concentration of cross-linker causes a progressive rise in the ratio of dimer to monomer for both wt (Fig. 6, lanes 1-3) and C129S/C131S (Fig. 6, lanes 4 -6). In the absence of the cross-linker, the surface form of the mutant receptor is mostly detected as monomer (Fig. 6, lane 4), whereas the dimeric species becomes the major form of the receptor with 5 mM BS 3 (Fig. 6, lane 6). This result suggests that the dimeric receptor is still the principal form present on the cell surface for C129S/C131S. In Fig. 6, twice as much volume of the immunoprecipitated samples in lanes 1 and 4 were loaded in lanes 2 and 5, whereas four times as much in lanes 1 and 4 were loaded in lanes 3 and 6. This increased loading is necessary because the inclusion of BS 3 reduces biotinylation for surface detection since both sulfo-NHS-Biotin and BS 3 form covalent bonds with the same pool of primary amines on the receptor.
To further demonstrate that C129S/C131S forms noncovalent dimers on the cell surface, we cotransfected a truncated C129S/C131S-containing receptor with the full-length C129S/ C131S-containing receptor, one of which is FLAG-tagged, and immunoprecipitated with anti-FLAG antibody (Fig. 7). If the nontagged and tagged receptors formed heterodimers, we would be able to coimmunoprecipitate the nontagged receptor with the tagged receptor. Since the monomeric full-length and cytoplasmic tail-truncated receptors can be resolved under reducing conditions on SDS-PAGE because of their differences in size, it should be possible to determine the relative amounts of the tagged and nontagged receptors on the cell surface using avidin as a probe for surface-biotinylated receptors on the blot. Fig. 7A, lane 7, shows that the nontagged truncated receptor, C129S/C131S/A877Stop, was coimmunoprecipitated in cotransfected cells with the FLAG-tagged full-length receptor, C129S/C131S, to a similar extent as the nontagged receptor, A877Stop, when it was cotransfected with FLAG-tagged wt (Fig. 7A, lane 2). If one of the molecular partners in cotransfected cells has C129S/C131S and the other does not, they apparently still associate with one another equally well. For instance, nontagged C129S/C131S/A877Stop can be coimmunoprecipitated with tagged wt (Fig. 7A, lane 3), and nontagged wt can be coimmunoprecipitated with tagged C129S/C131S/ A877Stop (Fig. 7A, lane 4). As a control, none of the nontagged receptors isolated from singly transfected cells could be immunoprecipitated by anti-FLAG antibody (data not shown).
In contrast to the results observed with cotransfection of A877Stop and wt (Fig. 7B, lane 2), a substantial amount of the coimmunoprecipitated complex, consisting of one or both of the C129S/C131S-containing receptors, can be dissociated by the addition of SDS sample buffer alone in the absence of reducing agents (Fig. 7B, lanes 3, 4, and 7). For instance, the coimmunoprecipitated complex of FLAG-tagged wt and nontagged C129S/C131S/A877Stop can be detected as a monomeric species in a 1:1 ratio (Fig. 7B, lane 3) under nonreducing conditions, even though most of the FLAG-tagged wt (Fig. 7B, lane 3) in the cotransfected cells is still detected as disulfide-linked homodimers, similar to that seen for wt transfected alone (Fig.  7B, lane 1). In other words, the monomeric species of wt observed in Fig. 7B, lanes 3 and 4 is actually associated noncovalently with C129S/C131S/A877Stop before the treatment with SDS sample buffer. The inclusion of iodoacetamide in the lysis buffer prevents the monomeric wt from associating with itself through disulfide linkages, specifically or nonspecifically, after it is dissociated from C129S/C131S/A877Stop in the SDSsample buffer. Thus, our results suggest that the CaR can form dimers through interactions other than intermolecular disulfide bonds.

Reconstitution through Heterodimerization of [Ca 2ϩ ] o -elicited [Ca 2ϩ ] i Responses in Cells Cotransfected with Two Inactive
Mutant CaRs, Both Carrying C129S/C131S-One test for dimerization of CaR monomers is the recovery of function observed for some inactive mutant pairs when they were cotransfected together in HEK293 cells (16). Similar rescue experiments were performed with mutant CaRs carrying C129S/ C131S, which were, therefore, incapable of forming covalently linked dimers. HEK293 cells were transiently cotransfected with pairs of mutant CaRs carrying one of the two inactivating mutations, E297K or A877Stop, with or without C129S/C131S. As a control, cells were also transfected with wt or mutant CaRs alone. CaR-mediated biological responses were examined by measuring [Ca 2ϩ ] o -elicited increases in [Ca 2ϩ ] i . As reported earlier (16), cells cotransfected with the individually inactive mutant CaRs, A877Stop and E297K, exhibited much greater responses than did cells transfected with either A877Stop or E297K alone. Introduction of the double mutations, C129S/ C131S, into E297K and A877Stop did not significantly alter the functions of the individual mutant receptors when transfected by themselves (Fig. 8, B and C; data are not shown for mutant receptors carrying A877Stop, which are completely inactive). Cotransfection of C129S/C131S/E297K with C129S/C131S/ A877Stop reconstituted CaR-mediated signaling to an extent almost identical to that observed in cells cotransfected with A877Stop and E297K (Fig. 8). Furthermore, Fig. 9 shows that C129S/C131S/E297K and C129S/C131S/A877Stop readily form heterodimers when cotransfected. For instance, nontagged C129S/C131S/A877Stop can be coimmunoprecipitated with FLAG-tagged C129S/C131S/E297K to an extent similar to that observed with nontagged A877Stop and tagged E297K (Fig. 9A,  lanes 3 and 4). However, a substantial amount of C129S/ C131S/A877Stop and C129S/C131S/E297K are not associated with one another through disulfide linkages (Fig. 9B, lanes 4  and 5), unlike A877Stop/E297K (Fig. 9B, lanes 3). As a control, the nontagged C129S/C131S/E297K and C129S/C131S/ A877Stop cannot be immunoprecipitated when transfected alone (Fig. 9, lanes 7 and 8). DISCUSSION The CaR principally exists as a dimer on the cell surface of CaR-transfected HEK293 cells, which is covalently linked by disulfide bonds (4). In previous attempts to identify the cysteines in the receptor extracellular domain responsible for CaR dimerization, two groups obtained discrepant results. Both groups identified two cysteines in the N-terminal extracellular domain that may be involved in intermolecular disulfide linkages, similarly to mGluR5 (2). However, one group proposed that Cys 101 and Cys 236 form intermolecular disulfide linkages, whereas the other showed that Cys 129 and Cys 131 mediate intermolecular disulfide linkages.
In the present studies, we initially focused on cysteines that are not very critical for receptor expression on the cell surface (15), assuming that the cysteines that participate in the intermolecular disulfide bonds are not critical for receptor protein folding. We found that mutating Cys 129 and Cys 131 , individually or in combination, has little effect on receptor protein expression. Consistent with the results of the study performed by Ray et al. (6), we found that the presence of the double mutations, C129S/C131S, eliminated most interomolecular di-sulfide linkages in the receptor proteins expressed on the cell surface. In contrast to the other study (5), however, we found that the double mutations, C101S and C236S, which interfere substantially with receptor expression when present either individually or in combination, do not disrupt disulfide-mediated dimerization.
The dimerization of many other G protein-coupled receptors, including the ␤ 2 -adrenergic receptor (9), the dopamine D2 receptor (17), the ␦ opioid receptor (3,18), and the bradykinin B 2 receptor (19), are not mediated by intermolecular disulfide bonds. For instance, constitutive dimerization of the ␤ 2 -adrenergic and the dopamine D receptors occurs via their transmembrane regions, whereas agonist-induced dimerization of the bradykinin B 2 receptor requires noncovalent interactions of the N terminus of the receptor. In this study, we have shown both biochemically and functionally that the removal of the intermolecular disulfide linkages normally present in the CaR does not affect its dimerization. As shown in Fig. 7A, lanes 2 and 7, pairs of CaRs with the double mutations, C129S and C131S, can associate with one another to an extent similar to those in which Cys 129 and Cys 131 are intact, even though the majority of the receptor species are no longer linked with one another through intermolecular disulfide bonds (Fig. 7B, lanes  3, 4, and 7). Functionally, coexpression of the inactive mutant CaRs, E297K and A877Stop, in which these double cysteine mutations were present also led to reconstitution of significant [Ca 2ϩ ] o -sensing and intracellular signaling capability, which was indistinguishable from that of cotransfected CaRs harboring E297K and A877Stop in which Cys 129 and Cys 131 were intact. One reason why others (6) did not find association of their truncated receptor, TM1 mutant, with C129S/C131S may be that the missing part in the TM1 mutant is likely essential for noncovalent association of dimeric receptors. This suggests that the CaR may form dimers through one or more noncovalent dimerization motifs present within the last six transmembrane domains and the cytoplasmic tail. Our current studies suggest that the cytoplasmic tail of amino acids 877 to 1078 is not essential for the noncovalent associations between our truncated and full-length receptors. Nevertheless, further studies are required to determine the structural domains mediating the noncovalent interactions with a focus on the region between TM5 and TM7, where putative hydrophobic dimerization domains exist.
We found that C129S, C131S, and C129S/C131S are all much more sensitive to [Ca 2ϩ ] o than wt. In addition, they all have reduced maximal responses in comparison to wt. C129S and C131S still form mostly intermolecular disulfide-linked dimers, suggesting that Cys 129 and Cys 131 are not only important for formation of intermolecular disulfide linkages but also for the normal function of the receptor even when the structural constraints conferred by formation of covalent bonds with another CaR molecule are still present. Consistent with the results of previous studies on several of the mutations causing autosomal dominant hypocalcemia, those mutations that are located in the vicinity of Cys 129 and Cys 131 , such as E127A and F128L, significantly increase the sensitivity of the CaR to [Ca 2ϩ ] o , even though they do not disrupt the formation of intermolecular disulfide bonds as suggested by Ray et al. (6).
In conclusion, our results support the involvement of Cys 129 and Cys 131 in sulfydryl-sensitive dimerization of the CaR and suggest that the region containing these two cysteines is important for the normal function of the CaR. However, the CaR still forms dimers, even in the absence of the intermolecular disulfide linkages mediated by Cys 129 and Cys 131 . Furthermore, CaRs lacking these disulfide linkages are still capable of the intermolecular interactions required for functional reconstitution of individually inactive mutant receptors, such as E297K and A877Stop.