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ABSTRACT G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences.

Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its

constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in

undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing

amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these

bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G

protein-coupled receptors. SIMILAR CONTENT BEING VIEWED BY OTHERS ASYMMETRIC ACTIVATION OF THE CALCIUM-SENSING RECEPTOR HOMODIMER Article 30 June 2021 PROMISCUOUS G-PROTEIN ACTIVATION BY THE

CALCIUM-SENSING RECEPTOR Article 17 April 2024 ALLOSTERIC MODULATION AND G-PROTEIN SELECTIVITY OF THE CA2+-SENSING RECEPTOR Article 07 February 2024 INTRODUCTION Cell surface receptors

generate intracellular signals upon activation by external stimuli. Like all proteins, these receptors exhibit constant conformational dynamics oscillating between inactive and active

states, which has been extensively studied for G protein-coupled receptors (GPCRs)1,2,3,4. Accordingly, receptors that maintain in an active state for sufficiently long periods can signal

without activation by external stimuli5,6,7,8,9. Such constitutive activity can leads to an optimal physiological output under evolutionary pressure, which is biologically significant10.

Indeed, many genetic mutations that have been found to either increase or decrease receptor constitutive activity can be the origin of human diseases6,11,12. For most GPCRs, the constitutive

activity can be easily detected6,13,14,15,16. However, some of them exhibit stable inactive conformations that lead to the undetectable constitutive activity, which permits receptors to

generate signals only upon agonist activation. For instance, α1B adrenergic receptor is well constrained in inactive state by a specific residue in the intracellular loop 317,18. In

addition, the inverse agonist cis-retinal stabilizes the rhodopsin in fully inactive state19. Conversely, some GPCRs exhibit strong constitutive activity that is almost equal to the activity

elicited by agonist stimulation. As an example, the melanocortin-4 receptor, in which endogenous ligands (agouti-related proteins) act as inverse agonists20. The constitutive activity can

be affected by the cellular environment. Indeed, constitutive activity of some metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, is naturally inhibited by the long form of the

intracellular partner Homer, but is revealed by the short form Homer1a21. Another example is the constitutive activity of 5-HT6 receptor, which is promoted by its interaction with

neurofibromin22 or Cdk523. GPCR constitutive activity can also be regulated by receptor phosphorylation24, dimerization25, alternative splicing26, or RNA editing27. The discovery of inverse

agonists and antagonists made it possible to identify the physiological significance of GPCR constitutive activity6,10. For example, Homer regulates the constitutive activity of mGluRs,

which is important for synaptic metaplasticity28,29, and the constitutive activity of histamine H3 receptor plays a leading role in regulating the activity of histamine neurons30. The class

C GPCRs that sense amino-acids and their derivatives31 usually exhibit significant constitutive activity21,32,33,34. However, that of the calcium-sensing receptor (CaSR)35 has not been well

studied. CaSR is a multifunctional receptor which plays important physiological role in organs involved or not involved in Ca2+ homeostasis36,37. The fundamental role of CaSR is to enable

parathyroid gland to detect subtle changes in extracellular Ca2+ concentration and to respond by modulating the release of parathyroid hormone (PTH) in the opposite direction38. The result

is Ca2+ normalization through the direct action of PTH, which promotes calcium release from bones and reabsorption by the kidney, in addition to its indirect effects in the gut. In the

parathyroid gland, CaSR signaling activity suppresses PTH secretion, thus preventing the development of hypercalcemia. This role of CaSR is also mediated by cell-autonomous activities in

kidney, bone and thyroid C cells. Consequently, if the receptor remains in a constant state of hyperactivity, it can lead to disturbances in calcium homeostasis and disorders such as

hypocalcemia. Many genetic mutations that lead to loss- or gain-of-function of the CaSR have been identified in patients with metabolic syndromes, such as familial hypocalciuric

hypercalcemia (FHH), neonatal severe primary hyperparathyroidism (NSPHT) or autosomal dominant hypocalcemia (ADH)37,39. In addition, CaSR autoantibodies that induced altered receptor

signaling properties have been identified in acquired hypocalciuric hypercalcemia40. Actually, three commercial drugs can target CaSR as positive allosteric modulator (PAMs).

Etelcalcetide41,42 targets the extracellular domain (ECD) and is applied in the treatment of secondary hyperparathyroidism, and two other drugs can bind to the transmembrane domain and are

used to the treat both primary and secondary hyperparathyroidism43,44. Like other typical class C GPCRs, such as metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid B receptor

(GABABR), GPRC6A and sweet/umami taste receptors, CaSR forms a constitutive dimer45. Each subunit within the dimer is composed of an extracellular Venus flytrap (VFT) domain, a cysteine-rich

domain (CRD) and a seven-transmembrane (7TM) domain. Among them, the VFT domain is responsible for the binding of calcium ions45 and L-AAs46 (Fig. 1a). Furthermore, the two protomers are

cross-linked by two pairs of disulfide bridges. Recently, the structure of full-length CaSR, both in inactive and active states with L-AAs, which shown to behave as pure-PAMs without direct

agonist effect (Fig. 1a), or with allosteric modulators, including etelcalcetide, have been analyzed47,48,49,50,51,52,53,54. It remains unclear the role of L-AAs in the constitutive activity

of CaSR. Here, we demonstrate that in contrast to other class C GPCRs, CaSR displays no detectable constitutive activity. We reveal that the covalent disulfide bridges between the two

subunits of this dimeric receptor are essential for the limitation of constitutive activity. More importantly, compare with the other class C GPCRs of the mGluR family that possess a single

inter-subunit disulfide bridge, CaSR has two disulfide bridges. Both two disulfide bridges are essential for blocking the constitutive activity and potential agonist effect of ambient L-AAs.

Several mutations of Cys residues that involved in these bridges had been identified in ADH patients. Interestingly, we found that all these mutations could generate constitutive activity,

which revealed the importance of the disulfide bridges in controlling calcemia. These data further illustrate the significant role of the inter-subunit disulfide bridge in class C GPCRs in

controlling their activity, and the importance of an evolutionarily optimized constitutive activity of receptors for proper physiological functions. RESULTS CONSTITUTIVE ACTIVITY OF CASR IS

NOT DETECTABLE We compared the constitutive activity of CaSR with mGluR5 by measuring the inositol phosphate-1 (IP1) accumulation, both of receptors are naturally coupled to the Gq protein.

As reported previously, a high constitutive activity of mGluR5 could be detected55 (Fig. 1b and Supplementary Fig. 1a). In contrast, the IP1 constitutive accumulation in CaSR transfected

HEK-293 cell was unable to be detected in the absence of calcium, even in the group with highest CaSR surface expression (Fig. 1b). Our data indicate that HEK-293 cells do not expressed

endogenous CaSR at a level high enough to be detected (Supplementary Fig. 1b, c). The constitutive activity of mGluR232, GABABR33,34, and GPRC6A56 also can be detected in transfected cells

as described in previous studies (Fig. 1c and Supplementary Fig. 1a). Co-transfected chimeric Gqi9 made it possible to detect the IP1 accumulation in HEK-293 cells that transfected with

mGluR2 and GABABR, for both receptors belong to the Gi/o coupled-receptors57. Altogether, these data showed that the CaSR activity is tightly controlled with a no detectable constitutive

activity toward the Gq pathway. UNDETECTABLE CONSTITUTIVE ACTIVITY OF CASR IS DUE TO THE INTER-SUBUNIT DISULFIDE BRIDGES To identify the molecular basis for the undetectable constitutive

activity in CaSR, we analyzed the numerous missense genetic mutations that have been reported to increase the activity of CaSR in patients with ADH disease39. Many mutations occurred in the

VFT domain (Fig. 1d), especially at the dimer interface that is known to control the structural dynamics and receptor activation2,4,47,58,59,60. Based on the inactive and active structures

of CaSR, there was reasonable to explain that the VFT dimer interface mainly consists of four regions48,51,58 (Fig. 1e). The upper loop contains a pair of cysteine residues (Cys129 and

Cys131) which were responsible for the covalent link between the two protomers, and was highly conserved in CaSR during evolution (Supplementary Fig. 2a). Interestingly, this upper loop

contains a hot spot of ADH mutations39. We therefore investigated the role of the inter-subunit disulfide bridges between the two upper loops (Fig. 2a). These Cys were replaced with Ser

since such natural mutations have been previously reported in ADH61,62. We found that C129S + C131S (CSCS) double mutations resulted in the appearance of monomer band in non-reducing

SDS-PAGE experiments, however, dimeric form of the mutant CSCS still could be detected on the cell membrane (Supplementary Fig. 2b, c). Indeed, dimers and even larger oligomers are observed

for many GPCRs in SDS-PAGE even when they are not covalently linked (i.e. many class A GPCRs). This is likely due to the consequence of unfolding by SDS and mixing the two chains during this

process, resulting in non-covalent dimers that are not sensitive to reducing agents such as DTT63. We further verified this result by bioluminescence resonance energy transfer (BRET) and

obtained a similar conclusion, which suggested that the dimerization of CaSR was maintained by multiple interactions (Supplementary Fig. 2d). Interestingly, both single and double mutants

displayed strong constitutive activity, despite their surface expression was similar to wild-type (WT) receptor (Fig. 2b and Supplementary Fig. 2e). The constitutive activity was positively

correlated with the cell surface expression of the mutated receptors (Fig. 2c) and could be blocked by the CaSR negative allosteric modulator NPS 214364 (Fig. 2d). The mutants also showed a

significant increase in CaCl2 potency compared with WT receptor (Fig. 2b). In addition, in contrast to the WT receptor, without agonist binding, the mutants became more sensitive in the

activation induced by PAMs, NPS R568 or AC265347, both of which bind to the 7TM domain of CaSR65,66 (Fig. 2e). Similar results were obtained by measuring the phosphorylated ERK1/2 (pERK),

these Cys mutants upregulated the basal level of pERK and NPS-2143 treatment inhibited this process (Supplementary Fig. 3a). Altogether, these results showed the absence of inter-subunit

disulfide bonds led to a high basal activity of CaSR. To further validate the correlation between inter-subunit disulfide bond and basal activity, we have re-built one inter-subunit

disulfide bond by changing the Ser132 into Cys in the CSCS construct. This mutant showed a similar dimerization pattern as the WT and single Cys mutants in non-reducing SDS-PAGE experiments

(Supplementary Fig. 3b). Interestingly, the CSCS + S132C construct showed a strong reduction of constitutive activity in both IP1 accumulation (Fig. 2f) and ERK phosphorylation when compared

with the CSCS construct (Supplementary Fig. 3c). Furthermore, addition of this S132C mutation in the CSCS construct could restore the Ca2+ pEC50 similar to the single Cys mutant

(Supplementary Fig. 3d). Of note, an another CaSR construct where both Cys129 and Cys131 were replaced by Ala also showed a similar increase in basal activity, that could be inhibited by

NPS2143 (Supplementary Fig. 3e). These results further indicated that the emergence of the constitutive activity in CaSR is a result of inter-subunit disulfide bridges removal. Taken

together, our data suggested that the inter-subunit disulfide bridges maintain the inactive state of CaSR and were required to restricting the basal activity of receptor. Interestingly, our

data also revealed that the presence of two disulfide bridges were crucial to the strong limitation of the basal activity, since a single mutation of Cys129 or Cys131 led to a moderate

constitutive activity (10–15% of the WT Emax; see Fig. 2b). And the double mutations that totally remove inter-subunit covalent link between VFTs generated a high constitutive activity (30%

of the WT Emax) (Fig. 2g). THE INTER-SUBUNIT DISULFIDE BRIDGE FAVORS THE BASAL ACTIVITY OF MGLURS Since the inter-subunit disulfide bridge is conserved in most class C homodimers, especially

in mGluRs (Fig. 3a), we wondered whether they play a similar role in controlling the basal activation. In contrast to CaSR, mGluRs have only one conserved Cys residue in the upper loop that

could establish single disulfide bridge (Fig. 3b). It was reported that mutated Cys121 to Ala in mGluR2 stabilized the VFT in inactive state4. Consistently, we found that the C121A mutant

has a lower constitutive activity in IP1 accumulation than the WT receptor when co-transfected with Gqi9 (Fig. 3c) and this decrease was not due to the difference in cell surface expression

(Supplementary Fig. 4a). In addition, to mimic the upper loop of CaSR and potentially favor the formation of two inter-subunit disulfide bridges, a second Cys residue was introduced at the

position 119 in mGluR2 equivalent to the first conserved Cys (Cys129) in the CaSR (Fig. 3c and Supplementary Fig. 4a, b). The H119C mutation significantly increased the basal activity of

mGluR2 (Fig. 3c) as well as its potency for glutamate (Fig. 3d). Similar experiments were also performed on mGluR5, which belongs to another group of mGluRs that are naturally coupled to the

Gq—phospholipase C signaling pathways. Similar to the mGluR2 mutants, deletion the inter-subunit disulfide bridge (C129A) in mGluR5 resulted in a lower basal activity, while an additional

Cys (V127C) could increase the basal activity (Fig. 3e and Supplementary Fig. 4c, d) and its potency for glutamate (Fig. 3f). Altogether, our results showed that the inter-subunit disulfide

bridge controls the basal activity of CaSR and mGluRs in distinct ways. REMOVAL OF INTER-SUBUNIT DISULFIDE BRIDGE ENHANCES SIGNALING OUTPUT OF CASR CaSR could respond to diverse ligands and

signals through different G proteins67,68. We investigated whether the CSCS mutant responds differently to these ligands and show preference toward different G proteins. As mentioned above,

the CSCS mutant was more sensitive to Ca2+ in comparison with WT CaSR, and the results from other ligands like Mg2+, neomycin and spermine show a similar effect of these ligands

(Supplementary Fig. 5a). To investigate the influence of this mutant on the activation of different Gq, Gi/o and G13 proteins, we measured the G protein dissociation by using BRET-based

sensors as previously described (Supplementary Fig. 5b)34,69. For all these G proteins, the BRET signal between the Gα and Gβγ subunits was lower in the CSCS group than WT under basal

conditions (Supplementary Fig. 5c–i). Upon agonist stimulation, the BRET signal measured for most G proteins showed increased pEC50 and decreased Emax except G13, which behaved differently

(Supplementary Fig. 5c–j). These results demonstrated that removal of the inter-subunit disulfide bridges in CaSR did not influence G protein selectivity under basal and agonist-induced

conditions. GENETIC MUTATIONS AT CYS129 AND CYS131 FAVOR THE CONSTITUTIVE ACTIVITY OF CASR Among the genetic gain-of-function mutations that are associated with ADH (Fig. 1d), several

mutations occurred at Cys129 or Cys131 (Fig. 4a)61,70. We tested the effect of six natural single mutations on the basal activity of CaSR and found that all these mutants showed a strong

basal activity (Fig. 4b) despite their surface expression was similar to WT receptor (Supplementary Fig. 6a). Furthermore, compared with WT receptor, these mutants showed higher Ca2+ potency

and more sensitive to NRS R568 (Fig. 4c–f). In addition, using the BRET-based G protein sensors, we showed that the mutant C131W, used as an example, has a higher basal activity in

different G protein pathways than WT receptor, and an increased pEC50 and decreased Emax in C131W also been revealed which were in consistence with the results obtained with the CSCS

construct. We also found that the CSCS construct showed higher sensitivity to CaCl2 compared to this C131W mutant except for the GoB pathway (Supplementary Fig. 6b–j). Therefore, the natural

mutations at either C129 or C131 would impair the integrity of the inter-subunit disulfide bridges and led to the basal activation of CaSR. L-AAS BINDING IN CASR IS REQUIRED FOR THE BASAL

ACTIVITY OF THE CSCS CONSTRUCT It was recently reported that ambient L-AAs in the cell culture medium were constitutively bound in the CaSR VFT (Fig. 5a), and act as pure-PAMs during

activation by extracellular Ca2+ 45,47,48,49,50,51,52,53. However, the extracellular concentrations of these L-AAs in cellular assays were difficult to control32,45, even in the absence of

serum. To study the importance of the L-AAs in the basal activity of the CSCS mutant, we constructed several single mutations that are known to prevent L-AAs binding in the VFT binding

pocket of CaSR45,47,49. Interestingly, the mutations abolished the constitutive activity of CSCS mutant (Fig. 5b and Supplementary Fig. 7a). Of note, the gain-of-function mutation E297D71

that was proposed to increase the affinity of L-AAs for CaSR45 had no effect on the basal activity of CSCS (Fig. 5b). Then we washed the cells three times with starvation buffer over 3 h to

remove the L-AAs that had bound to the receptor, and discovered that the basal activity of the mutations were significantly decreased in this group, and it could been recovered by the

addition of L-Trp (Fig. 5c). Elution and rebinding treatments with L-AAs showed that the spontaneous activity of the CSCS mutant was dependent on the intact amino acid binding. Moreover,

this agonistic effect of L-AAs was more significant with the CSCS + E297D construct, which is consistent with the higher sensitivity of the CSCS + E297D construct to L-AAs conferred by the

mutation E297D45. The results suggested that removal of the inter-subunit disulfide bridges altered the pure-PAM effect of L-AAs that become ago-PAMs. We further investigated whether the

binding of ambient L-AAs to both VFTs of the dimer was required to maintain the basal activity. We constructed a ‘controlled’ CaSR homodimer formed by the CaSRC1 and CaSRC2

subunits51,72,73,74. In these constructs, the C-terminus of the CaSR subunits were replaced by the C-terminus from modified GABAB1 (C1) or GABAB2 (C2) subunits, respectively. The dimers that

consisted of the same C-terminus (CaSRC1 + CaSRC1 or CaSRC2 + CaSRC2) hardly reached the cell surface (Fig. 5d and Supplementary Fig. 7b) and responded weakly to the CaCl2 stimulation

(Supplementary Fig. 7c). Then we introduced a S170A single mutation to the CSCSC1-CSCSC2 constructs. This mutation that occurs in CSCSC1 or CSCSC2 alone strongly decreased the basal

activity, and more importantly, when S170A mutation is in both VFTs it abolished the constitutive activity (Fig. 5e and Supplementary Fig. 7d). These results suggested that L-AA binding in

at least one VFT of CSCS was required to generate constitutive activity, and binding in both VFTs could produce the strongest basal activity (Fig. 5f). Altogether, these data showed the

importance of L-AAs induced VFT closure in the basal activity of CSCS. UPPER INTERFACE OF THE VFT IS NOT INVOLVED IN CASR BASAL ACTIVITY In addition to the inter-subunit disulfide bridges,

we wondered whether other regions at the VFT interface could control the constitutive activity of CaSR. Other direct interactions between the two LB1 were the extended loops which could

stabilized the VFT interface through hydrophobic and polar interactions (Fig. 1e and Supplementary Fig. 8a). This unique structure is not conserved in the other class C GPCRs (Supplementary

Fig. 8b). Two loss-of-function genetic mutations in this loop, S53P and P55L39,70 (Supplementary Fig 8a) showed no effect on the basal activity of the receptor (Supplementary Fig. 8c, d).

But both mutants have a lower potency for calcium activation (Supplementary Fig. 8e), which were in line with their classification as inactivating mutations. In addition, we analyzed another

region of the LB1 interface that is conserved in the other class C GPCRs (Supplementary Fig. 8f, g). The two loss-of-function genetic mutations in this region, L159F and L159R75,76, as well

as L108A, L112A, and L156A had no effect on the basal activity of receptor (Supplementary Fig. 8h, i), but decreased the pEC50 of Ca2+ (Supplementary Fig. 8j). On the other hand, the

residue Phe160 located immediately after helix C, which may interact directly with the upper loop (residue Val115) in both the inactive and active states (Supplementary Fig. 8k), was also

mutated. Interestingly, the F160A mutant shows a significant constitutive activity (Supplementary Fig. 8l) and higher Ca2+ potency than the WT (Supplementary Fig. 8j). This is probably due

to an effect on the conformation of the upper loop via the loss of interaction with the side chain of Val115, which could lead to its repositioning. Indeed, Val115 is located between the end

of helix B and the beginning of the upper loop that adopts a helical conformation, a critical position for the relative positioning of helix B and the upper loop (Supplementary Fig. 8k).

Altogether, our data showed that the LB1 interface of the VFTs, apart from the upper loop containing the disulfide bridges, is not involved in CaSR basal activity. NEGATIVE CHARGES IN THE

LOWER INTERFACE OF THE VFT LIMIT CASR ACTIVATION BUT IS NOT INVOLVED IN BASAL ACTIVITY In contrast to mGluRs and other class C GPCRs, the lower interface of the CaSR VFT (LB2) was highly

enriched with negatively charged residues (Fig. 6a, b). Our recent study have ruled out the possibility that this region has a functional binding site for Ca2+ 45. To investigate the effect

of LB2 interface on basal activity, we constructed a mutant where all the 13 negatively charged residues in the acidic patch been replaced by alanine, and we named this mutant 13 A (Fig. 6c,

Supplementary Fig. 9a). The 13 A mutant showed no effect on basal activity when made in the WT receptor, while the introduction of 13 A mutation in the CSCS construct further increased the

basal activity (Fig. 6d). In addition, the similar pEC50 for Ca2+ indicated that introduction of 13 A mutations did not impair the activation of CaSR by Ca2+, both in WT and CSCS constructs

(Fig. 6e), which was consistent to the results obtained with single or multiple mutants in this acidic LB2 patch in previous study45. Interestingly, the 13 A mutations potentiated the

activation of CaSR by NPS R568, both in WT and CSCS constructs (Fig. 6f). Finally, we analyzed the effect of the PAM etelcalcetide, which could form a disulfide bond with the free LB2

Cys48242 and binds to the LB2 interface by forming salt bridges with the patch of negatively charged residues51 (Supplementary Fig. 9b). Etelcalcetide showed no impact on the basal level of

IP1 accumulation in WT receptor, but elevated the basal activity of the CSCS construct (Supplementary Fig. 9c). Moreover, etelcalcetide could increase the Ca2+ potency of WT and CSCS mutant

(Supplementary Fig. 9d), as well as the agonist effect of NPS R568 on both WT and CSCS constructs (Supplementary Fig. 9e). Altogether, our results showed that the negatively charged

interface of LB2 exerted no effect on constitutive activity of WT receptor but could enhance it in the CSCS construct, underscoring the pivotal role of inter-subunit disulfide bonds in

limiting the constitutive activity of CaSR. It also suggests that when both LB2s could come closed, it facilitates the activation of CaSR by PAM binding in the 7TM. DISCUSSION As a special

nutrient-sensing receptor regulated by various ligands, CaSR develops a unique ability to sense the tiny fluctuation of extracellular Ca2+ 77,78. This response is essential for tight control

of calcemia through various mechanisms, including in the parathyroid which controls PTH release, in the bones and kidney among others37. Therefore, the CaSR activity was critical for proper

physiological response. In this study, we showed that inter-subunit disulfide bridges functions as molecular locks to significantly restrict the constitutive activity. They also reduce

receptor sensitivity to the agonists, as well as inhibit the agonism of CaSR by natural and synthetic PAMs, thus setting the Ca2+ potency to an optimal value. Class C GPCRs are complex

GPCRs79, the majority of them have constitutive activity34,55,79,80,81. Their activation mechanism involves several steps, such as the closure and reorientation of VFT, which leads the CRD

closed to each other, followed by a direct interaction between the 7TM domains through TM6 and then a conformational change in one of the 7TM domain60. Consequently, their constitutive

activity can originate from various structural elements. First, as demonstrated in GABABR, it could come from the spontaneous closure of VFT, which is abolished by the competitive

antagonist33,59. Alternatively, it could also originate from any other step following the closure of the VFT such as the CRD and 7TM dimer rearrangement82. As demonstrated in mGluR5, the

constitutive activity could come from the 7TM domain that can reach an active state even if the VFTs remain in their inactive orientation55. GABABR is an additional instance where a genetic

mutation S695I in human GABAB2 7TM could trigger strong constitutive activity, which was associated with epileptic encephalopathy63. As such, several specific structural determinants in the

CaSR could be largely limit or even suppress its constitutive activity. This is in line with the identification of more than one hundred mutations in the ECD and 7TM domains of CaSR in

patients with gain-of-function CaSR associated with ADH39. However, only a few of them, such as N802I in TM6 and A843E in TM7, have been investigated for their constitutive activity83,84. In

the present study, we examined the possible role of the subunit interface at the level of the VFT dimer, as it may regulate the basal activity of CaSR. We show that the inter-subunit

disulfide bridges on the upper loops of LB1 is required to maintain a completely inactive state with low Ca2+ potency in the 2.2–2.6 mM range. This concentration range is optimal for the

physiological function of the receptor in controlling calcemia. We uncovered that all natural mutations occurring at either C129 or C131 could trigger the constitutive activation of CaSR.

Therefore, the integrity of two inter-subunit disulfide bridges is required to abolish constitutive activity of CaSR. This finding might provide an explanation for calcemia induced by

mutations in either C129 or C131. Constitutive internalization and constitutive activity are usually related, and could be blocked by reverse agonists. Previous studies demonstrated that

CaSR undergoes constitutive and agonist induced internalization, both of which were G protein-independent and β-arrestin-dependent at least partially85. Interestingly, a negative allosteric

modulator did not inhibit the constitutive internalization of CaSR, which suggests there is no link between basal activity and constitutive internalization of CaSR. However, it needs to be

examined in more details in future studies. Among the class C GPCRs examined in this study, only CaSR had no detectable constitutive activity, this observation may be attributed to the fact

that CaSR has two inter-subunit bonds. These cysteine residues have been conserved from fish to mammals, illustrating their evolutionary importance and their ability to tightly control a

very low basal activity. In contrast to what was observed for CaSR, the mGluRs and GPRC6A which only have one inter-subunit bridge, mutation of the unique inter-subunit disulfide bond in

mGluR2 reduced the apparent agonist affinity and decreased the stability of the active state4. The distinct roles of inter-subunit disulfide bonds between mGluRs and CaSR may be due to the

different sequence and conformation of the upper loop where the disulfide bridge is located, but also the preceding α-helix B which participates in the dimer interface between the two

protomers. In mGluRs, this helix B adopts a different conformation between the inactive and active states. Interestingly, the loop immediately preceding it contains residues important for

ligand recognition in the VFT binding pocket86. Agonist binding in the VFT can therefore trigger relaxation of the inter-subunit interface by acting on this helix B, which releases its

constraint upon activation. Concerning the upper loop, its structure is often not solved in the class C GPCRs because of its flexibility51,87,88,89. However, in the few structures where this

loop could be observed, its conformation in CaSR and mGluRs is different (Supplementary Fig. 10a–d). Another difference in the activation mechanism between CaSR and mGluRs, which could

affect constitutive activity, is the large amplitude of VFT dimer reorientation observed for mGluRs between inactive and active states. In CaSR, the amplitude of VFT reorientation is not as

large as in the mGluRs. Mutation of the inter-subunit disulfide bridge in mGluRs could limit this VFT reorientation, thus reducing constitutive activity as observed in our study for both

mGluR2 and mGluR5. In brief, it is reasonable to presume that the inter-subunit disulfide bond in the upper loop may affect the receptor’s equilibrium between active and inactive state1,2,

but additional experimental approaches and methods are required to fully investigate the precise mechanisms in the future. Our findings with inter-subunit disulfide bridge mutants provide

insights on the molecular mechanism of CaSR activation. Cryo-EM structures of CaSR confirmed that L-AAs binding stabilizes the closed state of VFT, an intermediate state which is more easily

activated by Ca2+ but does not result in receptor activation49. We uncovered through mutagenesis that the basal activity induced by impairing the inter-subunit disulfide bond was most

probably caused by the ambient L-AAs. And when the integrity of the inter-subunit covalent link was lost, L-AAs occupied only one VFT pocket were sufficient to drive the receptor activation.

Removing the inter-subunit covalent link most likely decreases the activation energy barrier and enabling L-AAs binding to activate the receptor, as well as increasing the agonist potency

of the receptor. This further validates the function of inter-subunit disulfide links in restricting the CaSR activation for better adaptation to complex environmental stimuli, and also

explains why L-AAs have no agonist activity but behave as pure-PAMs45. Our results on the LB2 interface also help to clarify the molecular mechanism of CaSR activation. This region with

highly negatively charged residues is involved in the contact formation between LB2 domains upon the activation of class C GPCRs. Binding of cations, such as Gd3+, can neutralize the

electrostatic repulsion in mGluRs and stabilize receptor active conformation90. The CaSR LB2 interface exhibits significantly higher electrostatic repulsion than mGluRs because it contains

approximately twice as many negatively charged residues45,58. The electrostatic repulsion from this region was proposed to be able to allosterically control CaSR activation58. Our previous

study demonstrated that mutations in this region did not alter the Ca2+ potency and excluded the possibility that this region has a functional binding site for Ca2+ 45. Here we show that

neutralization of negatively charged residues in this region fails to change the Ca2+ potency, but significantly enhanced the agonist effect of PAMs on CaSR and the basal activity of the

CSCS mutant. Therefore, we speculated that the inter-subunit covalent bridges has a stronger importance than the electrostatic repulsion caused by the LB2 interface to stabilize the inactive

state. Disruption of the inter-subunit disulfide bridges could bring the two LB2 lobes into proximity to induce an activation signal. And this activation signal could be further facilitated

by reducing the electrostatic repulsion at the LB2 interface. However, in presence of the inter-subunit disulfide bridges, only reducing the electrostatic repulsion in the receptor did not

alter the calcium potency or basal activity. Overall, inter-subunit disulfide bridges are the main determinant limiting receptor activation by raising the energetic barrier to CaSR

activation. Electrostatic repulsion at the LB2 interface is a secondary determinant that reinforces stabilization of the inactive state. The combination of these two determinants enables

CaSR to accurately detect surrounding complex nutrient signals. Taken together, our study demonstrates that the specific structural framework of VFT provide molecular basis for the unique

activation features of CaSR, permitting for fine tuning of the ligands sensitivity of the receptor. All results allowed us to propose a molecular lock model, which speculates that the

inter-subunit disulfide bonds serve as molecular locks to restrict the receptor’s dynamics and stabilize it in inactive conformations (Fig. 7). Removal of the inter-subunit disulfide bonds

enables L-AAs alone to activate CaSR and elevates the receptor’s agonist potency. Therefore, these findings highlight the importance of optimal constitutive activity of receptors and provide

valuable insights for the design and development of targeted drugs. METHODS MATERIALS Calcium chloride (Cat. 10043-52-4), magnesium chloride (Cat. 7786-30-3), GABA (Cat. A2129), L-glutamate

(Cat. 56-86-0), L-alanine (Cat. 56-41-7), L-trytophan (Cat. 73-22-3), neomycin (Cat. 1405-10-3) and spermine (Cat. 71-44-3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). NPS R-568

(Cat. 3815) and AC265347 (Cat. 6165) were purchased from Tocris Bioscience (Bristol, UK). NPS 2143 (Cat. ab145050) was obtained from Abcam (Cambridge, UK). Etelcalcetide (Cat. HY-P1955A)

was from MedChemExpress (NJ, USA). Lipofectamine 2000 (Cat. 11668019) and Fluo4-AM (Cat. M14206) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). SNAP-Surface 649 (Cat. S9159S)

was from New England Biolabs (Ipswich, MA, USA). IP-One Gq kit (Cat. 62IPAPEB), BRET substrate coelenterazine h (Cat. S2001) and furimazine (Cat. N1120) were from Revvity (Codolet, France)

and Promega (Madison, WI, USA), respectively. PLASMIDS AND TRANSFECTION All the plasmids encoding the receptors CaSR, mGluR2, mGluR5, and GPRC6A have a SNAP-tag before the HA- or Flag-tag.

The pRK5 plasmid encoding wild-type human CaSR, with Flag or HA and SNAP tags inserted after the signal peptide45. We have verified that the Flag and HA-tagged CaSR are similarly expressed

on the cell surface (Supplementary Fig. 11a) and showed they have the same potency and Emax upon calcium stimulation (Supplementary Fig. 11b), under the same transfection conditions. The

pRK5 plasmid encoding the rat mGluR2 or mGluR5, labeled with HA and SNAP inserted just after the signal peptide73,82. The pRK5 plasmid encoding rat GB1, tagged with HA inserted after the

signal peptide, and the rat GB2, tagged with Flag inserted after the signal peptide were kindly provided by the Institut de Génomique Fonctionnelle (Montpellier, France)63. The pRK5 plasmid

encoding wild-type human GPRC6A, with a Flag and SNAP tags inserted after the signal peptide was a gift from Revvity. For BRET saturation experiments, plasmids were generated by inserting

the Rluc or YFP sequence at the C-terminus of Flag-SNAPCaSR. The last 192 residues of CaSR C-terminus were replaced by the sequence coding C1KKXX (C1, the coiled-coil sequence of 47 residues

at the C-terminus of GABAB1) or C2KKXX (C2, 49 residues of GABAB2 coiled-coil region, followed by the endoplasmic reticulum retention signal KKTN) to obtain the plasmids encoding

HA-SNAPCaSRC1 and Flag-SNAPCaSRC2. The mutations for CaSR and mGluRs were generated by site-directed mutagenesis using the QuikChange mutagenesis protocol (Agilent Technologies). All

constructs were verified by DNA sequencing. All experiments were performed in HEK293 (ATCC, CRL-1573, lot: 3449904) cultured in DMEM media (Thermo Fisher Scientific) containing 10% fetal

bovine serum (Thermo Fisher Scientific), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37 °C and 5% CO2. Cells were transiently transfected either by

electroporation or using lipofectamine 2000 according to the manufacturer protocol (Invitrogen Life Technologies)72. In the electroporation transfection, ten million cells were transfected

with a total of 4 μg plasmids. In the liposome transfection assay, two million cells were transfected with 1 μg of total amounts plasmid of interest. In order to couple mGluR2 and GABABR to

the phospholipase C pathway, the cells were co-transfected with the chimeric G protein Gqi9. For experiments involving mGluRs, the cells were also co-transfected with the glutamate

transporter EAAC1. The ratio of amount plasmids for the constructs expressing the indicated receptor, Gqi9 and EAAC1 is 2:1:1. ENZYME-LINKED IMMUNOSORBENT ASSAY Cell surface and total

expression of the indicated constructs were quantified by an enzyme-linked immunosorbent assay (ELISA). Cell culture medium was removed from 96-well plates 24 h after transfection, then

cells were washed and fixed with 4% paraformaldehyde (Sigma-Aldrich), then blocked with 10% FBS (for the total expression group, 0.1% Triton X-100 (Thermo Fisher Scientific) was required

before this step to increase the permeability of the cell membrane). Flag-tagged constructs were detected with the monoclonal mouse anti-Flag antibody coupled with horseradish peroxidase

(1:20,000; F1804, Sigma-Aldrich). HA-tagged constructs were detected with the rat anti-HA antibody coupled with horseradish peroxidase (1:20,000; 3F10, Roche, Indianapolis, USA). Bound

antibodies were detected by chemoluminescence using a Super Signal substrate (Thermo Fisher Scientific) and a 2103 EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).

INTRACELLULAR CALCIUM MEASUREMENTS Intracellular Ca2+ release was measured in 96-well plates of HEK293 cells with a calcium-sensitive fluorescent dye (Fluo4-AM, Thermo Fisher Scientific)45.

Briefly, 24 h after transfection, cells were washed and pre-incubated with 1 μM Fluo-4 AM in Flex buffer (containing 130 mM NaCl, 5.1 mM KCl, 0.42 mM KH2PO4, 0.32 mM Na2HPO4, 5.27 mM

glucose, 20 mM HEPES, 3.3 mM Na2CO3, 0.1% BSA, 2.5 mM probenecid, pH 7.4) at 37 °C for 1 h. Then, cells were washed once with Flex buffer and 50 μl of this buffer was added into the wells.

And 50 μl of the indicated compounds at 2-fold final concentrations were injected at 20 s during 60 s recording. Fluorescence signals (excitation 485 nm, emission 525 nm) were measured by

using the fluorescence microplate reader Flexstation 3 (Molecular Devices, Sunnyvale, CA, USA). The Ca2+ response is given as the agonist-stimulated fluorescence increase. INOSITOL PHOSPHATE

(IP1) MEASUREMENTS IP1 accumulation was determined in 96-well plates using the IP-One HTRF kit (62IPAPEJ, Revvity) according to the manufacturer’s instructions. Briefly, 24 h after

transfection, cells were washed and incubated with indicated compounds in the Ca2+-free stimulation buffer (containing 10 mM HEPES, 146 mM NaCl, 4.2 mM KCl, 1 g/L glucose, 50 mM LiCl, pH

7.4) at 37 °C for 30 min. The d2-labeled IP1 (IP1-d2) and terbium cryptate-labeled anti-IP1 antibody (Anti-IP1-K) were diluted in lysis buffer (provided by the IP-One HTRF kit) and added in

each well, then the plate was incubated in the dark at room temperature for 1 h before detected by the Multi-mode plate reader (PHERAstar FSX, BMG LABTECH). Results were calculated from the

standard curve in the kit with the fluorescence ratio of IP1-d2 emission at 665 nm over the Anti-IP1-K emission at 620 nm. For the experimental group of L-AAs elution and rebinding

treatments, the Mg2+ and Ca2+ free HBS starvation buffer (containing 10 mM HEPES, 140 mM NaCl, 4 mM KCl, 1 mM KH2PO4, pH 7.4) was used to wash cells for three times during 3 h, then the

cells were incubated with the indicated compounds in the stimulation buffer. BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET) To measure the dimerization of CaSR WT and the indicated

mutants, two million cells were co-transfected with a constant amount of Rluc-tagged receptors (0.2 μg) and an increasing amount of YFP-tagged receptors (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8,

and 1 μg) and completed to a total amount of 1.2 μg with the plasmid encoding the pRK5 empty vector. Cell culture medium was removed from 96-well plates 24 h after transfection, then cells

were washed and incubated in HBS buffer (containing 10 mM HEPES, 140 mM NaCl, 4 mM KCl, 1 mM KH2PO4, pH 7.4) at 37 °C. YFP fluorescence was measured before the addition of Rluc substrate

coelenterazine H. Then each well was loaded with 40 μL HBS with coelenterazine H at a final concentration of 5 μM, and the signal was read by a Mithras LB 940 (Berthold Biotechnologies, Bad

Wildbad, Germany) at 37 °C for six cycles. The net BRET was defined as the experimental BRET signal values with the baseline subtracted. For G protein activation experiments, two million

cells were transfected with 0.5 μg receptor plasmid, 0.02 μg the indicated Gα fused to Nluc, 0.2 μg Gβ1 and 0.2 μg Venus-tagged Gγ9 by Lipofectamine 2000. Cell culture medium was removed

from 96-well plates 24 h after transfection, then cells were washed and each well was loaded with 40 μL of the Nluc substrate furimazine at a final concentration of 10 μM. After measuring

the baseline BRET signal, cells were treated with 10 μL of drugs (prepared in HBS at 5-fold final concentration) for an additional 6 cycles. Results were calculated as the ratio of the Venus

emission at 485 nm over the Nluc emission at 530 nm. WESTERN BLOT Twenty-four hours after transfection, cells were washed and incubated with or without the indicated treatments in HBS

buffer at 37 °C for 2 h. Then the cells were lysed with ice-cold lysis buffer (containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS), supplemented

with complete phosphatase inhibitor cocktail (Roche) and incubated on ice for 30 min. Samples were denatured with loading buffer (containing 250 mM Tris-HCl pH 6.8, 50% (v/v) glycerol, 10%

(w/v) SDS, 0.5% (w/v) bromophenol blue) and 10 mM DTT for 10 min at 95 °C, separated by SDS-PAGE (10% w/v), and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA)

and washed with blocking buffer (5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20) for 2 h at 25 °C. The following primary antibodies were used for western blot analysis:

polyclonal anti-p44/42 ERK and anti-phospho-p44/42 ERK antibodies (1:3000; 9101 and 9102, Cell Signaling Technology, Shanghai, China), monoclonal Flag antibody (1:1000; KM8002, Sungene

Biotech, Tianjin Province, China) and polyclonal anti-α-tubulin antibody (1:3000; KM9007, Sungene Biotech, Tianjin Province, China). The primary antibodies were incubated overnight at 4 °C,

followed by incubation with the DyLight 800 4 X PEG-conjugated secondary antibody (1:20,000; 5151 and 5257, Cell Signaling Technology, Shanghai, China) for 2 h at 25 °C. The specific protein

bands were visualized by Odyssey CLx imager (LI-COR Bioscience, Lincoln, NE, USA). The density of bands was measured by ImageJ software (Bethesda, MD, USA). FLUORESCENT-LABELED BLOT

EXPERIMENTS The dimerization pattern of CaSR mutants were measured by fluorescent-labeled blot57,63. Briefly, 24 h after transfection, adherent HEK293 cells in 12-well plates were incubated

with 100 nM SNAP-Surface 649 in culture medium in the dark at 37  °C for 1 h, and then lysed with lysis buffer (containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium

deoxycholate, 0.1% SDS) for 1 h at 4 °C. After centrifugation at 12,000 × _g_ for 30 min at 4 °C, the supernatants were mixed with loading buffer (containing 250 mM Tris-HCl pH 6.8, 50%

(v/v) glycerol, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue) at 37 °C for 10 min and separated by SDS-PAGE (8% w/v). In reducing conditions, samples were treated with 100 mM DTT in loading

buffer for 10 min before loading the samples. Proteins were transferred to nitrocellulose membranes (Millipore). The specific protein bands were visualized by Odyssey CLx imager (LI-COR

Bioscience). REVERSE TRANSCRIPTION PCR AND REAL-TIME PCR Total RNA of mock and CaSR-overexpressed cells were extracted using Trizol reagent (Invitrogen, Carlsbad, USA) and reverse

transcribed into cDNA using the Superscript first-strand synthesis system (Invitrogen). _GAPDH_ and _CASR_ mRNA expressions were examined. The primers used were as follows: _GAPDH_

(NM_001256799), 5′-tcaccagggctgcttttaacc-3′ and 5′-gacaagcttcccgttctcag-3′; _CASR_ (NM_000388), 5′-ccctctacgattgctgtggt-3′ and 5′-agtctgctggaggaggcata-3′. Real-time PCR was conducted using

the SYBR Green (Vazyme Biotechnology, Nanjing, China) and the StepOne plus Real-time PCR System (Thermo Fisher Scientific). The threshold was set according to the exponential phase of

products, and the cycle threshold (CT) value for samples was determined. The resulting data were analyzed with the comparative CT method for relative gene-expression quantification against

_GAPDH_. MOLECULAR MODELING The structures of the receptors involved were analyzed and displayed with PyMol software (Palo Alto, CA, USA). The molecular models were generated with PyMol

software based on the structures from the PDB (7DTV, 7DTW, 5FBK, 7M3E, 7M3G, 7M3J and 7M3F for CaSR, 7EPA and 7EPB for mGluR2, 7FD8 for mGluR5). The sequence of CaSR between different

species or the human CaSR and different rat mGluR subtypes were aligned with Clustal Omega and displayed by ESPript 391. STATISTICS AND REPRODUCIBILITY Data were analyzed with Prism 7

software (GraphPad Software, San Diego, CA, USA). Concentration-responses curve parameters were derived using a four parameters non-linear regression equation. Unless stated otherwise, data

shown in the figures represent the mean ± S.E.M. of at least three independent experiments performed in triplicates. Statistical differences were determined by GraphPad Prism using one-way

ANOVA with a Dunnett’s multiple comparison test, two-way ANOVA with Sidak’s multiple comparisons test or t test. _P_ < 0.05 was considered to be statistically significant. DATA

AVAILABILITY Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. The source data underlying the graphs in the manuscript are

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(2018). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported by grants from National Key R&D Program of China (grant numbers 2022YFE0116600

and 2021ZD0203302 to J.L.), National Natural Science Foundation of China (NSFC) (grant numbers 32330049 and 82320108021 to J.L., 31070737 and 31371423 to P.Y.), Key Technologies R&D

Program of Guangdong Province (grant number 2010A080813001 to J.L.). P.R. was supported by Fondation pour la Recherche Médicale (EQU202303016470), the Institut National de la Santé et de la

Recherche Médicale (INSERM; International Research Program «Brain Signal») and the Franco-Chinese Joint Scientific and Technological Commission (CoMix) from the French Embassy in China.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cellular Signaling Laboratory, International Research Center for Sensory Biology and Technology of MOST, Key Laboratory of Molecular Biophysics

of MOE, and College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China Shumin Ma, Xueliang Yin, Ping Yi & Jianfeng Liu * Institut de

Génomique Fonctionnelle (IGF), Université de Montpellier, CNRS, INSERM, Montpellier, Cedex 5, France Jean-Philippe Pin & Philippe Rondard Authors * Shumin Ma View author publications You

can also search for this author inPubMed Google Scholar * Xueliang Yin View author publications You can also search for this author inPubMed Google Scholar * Jean-Philippe Pin View author

publications You can also search for this author inPubMed Google Scholar * Philippe Rondard View author publications You can also search for this author inPubMed Google Scholar * Ping Yi

View author publications You can also search for this author inPubMed Google Scholar * Jianfeng Liu View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS S.M. and P.Y. designed research; S.M. and X.Y. performed research; S.M., P.Y., J-P.P., and P.R. analyzed data; and S.M., P.Y., J-P.P., P.R., and J.L. wrote the paper.

CORRESPONDING AUTHORS Correspondence to Philippe Rondard, Ping Yi or Jianfeng Liu. ETHICS DECLARATIONS COMPETING INTERESTS Philippe Rondard and Jean-Philippe Pin are involved in a

collaborative team between the CNRS and Revvity (IGF, Montpellier). All other authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Communications Biology_ thanks

Caroline Gorvin, Jan Voss, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Isabelle Lucet and David Favero. ADDITIONAL

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ma, S., Yin, X., Pin, JP. _et al._ Absence of calcium-sensing receptor basal

activity due to inter-subunit disulfide bridges. _Commun Biol_ 7, 501 (2024). https://doi.org/10.1038/s42003-024-06189-3 Download citation * Received: 21 August 2023 * Accepted: 12 April

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