Extracellular calcium ions (Ca²⁺) are required for various kinds of biological processes in the human body. Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that senses small fluctuations of extracellular levels of Ca²⁺ ions in the blood (Brown et al., 1993). It maintains Ca²⁺ homeostasis by the modulation of parathyroid hormone (PTH) secretion from parathyroid cells and the regulation of Ca²⁺ reabsorption by the kidney (Brown, 2013). Recently, it has been reported that CaSR is also a phosphate sensor that can sense moderate changes in extracellular phosphate concentration (Centeno et al., 2019; Chang et al., 2020; Geng et al., 2016). Dysfunctions of CaSR or mutations in its genes may lead to Ca²⁺ homeostatic disorders, such as familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia (Hendy et al., 2009; Pollak et al., 1993; Ward et al., 2012).

CaSR belongs to the family C GPCR that includes gamma-aminobutyric acid B (GABA_B) receptors, metabotropic glutamate receptors (mGluRs), taste receptors, GPRC6a, and several orphan receptors (Ellaithy et al., 2020; Hannan et al., 2018; Heaney and Kinney, 2016; Pin and Bettler, 2016). Like most class C GPCRs, CaSR functions as a disulphide-linked homodimer. Each subunit of CaSR is comprised of a large extracellular domain (ECD) that contains a ligand-binding Venus flytrap (VFT) domain and a cysteine rich domain (CRD), and a seven-transmembrane domain (7TMD) that connects to CRD to carry signals from VFT domain to downstream G proteins (Geng et al., 2016; Zhang et al., 2016).

CaSR can be activated or modulated by Ca²⁺ ions, amino acids (Geng et al., 2016; Liu et al., 2020; Zhang et al., 2016), L-1,2,3,4-tetrahydronorharman-3-carboxylic acid (TNCA), a tryptophan derivative ligand (Zhang et al., 2016), and several commercial calcium mimetic drugs, such as cinacalcet (Leach et al., 2016; Nemeth et al., 2004), etelcalcetide, and evocalcet (positive allosteric modulator, PAM, of CaSR) that are used for patients with end-stage kidney diseases undergoing dialysis (Alexander et al., 2015; Leach et al., 2016; Walter et al., 2013).

Recent groundbreaking structural studies of several full-length class C receptors, such as mGluR5 (Koehl et al., 2019) and GABA_B receptors (Kim et al., 2020; Mao et al., 2020; Papasergi-Scott et al., 2020; Park et al., 2020; Shaye et al., 2020), by cryo-electron microscopy (cryo-EM) have provided a structural framework to unravel the activation mechanisms of class C GPCRs. The crystal structures of the resting and active conformations of CaSR ECD were solved by two different groups (Geng et al., 2016; Zhang et al., 2016). More recently, Ling et al. have solved the cryo-EM structures of full-length CaSR in active and inactive states; however, their inactive structures do not show the fully inactive state and exhibit some characteristics of the active conformation of crystal CaSR ECD (Ling et al., 2021; Geng et al., 2016). In their active structures, they proposed that Ca²⁺ ions and L-Trp work cooperatively to activate CaSR, leading to the closure of VFT domain.

In our study, we used cryo-EM to obtain the structures of full-length CaSR in inactive and agonist+PAM bound conformations. The fully inactive structure is stabilized by a negative allosteric nanobody. In the agonist+PAM bound structure, we identified a new calcium binding site at the interdomain cleft of VFT, with Ca²⁺ and TNCA constitute a composite agonist to stabilize the closure of the VFT, leading to the conformational changes of the 7TMDs to initiate signaling.

In this study, we have determined the cryo-EM structures of CaSR in the fully inactive and agonist+PAM bound states. During the preparation of our manuscript, several CaSR structures have been reported, including structures of closed–closed conformation in the inactive state (CaSR^Icc and CaSR^Trp) and closed–closed conformation of Ca²⁺/Trp bound state (CaSR^Acc and CaSR^Ca). Open-closed and open–open conformations (CaSR^Ioc and CaSR^Ioo) have also been observed; however, they were not built due to low resolution (Ling et al., 2021). The overall conformation of our CaSR^agonist+PAM structure is almost identical to that of CaSR^Acc, while the open–open conformational VFT module in our inactive structure (CaSR^{fully inactive}) is different from the closed–closed conformation in their reported inactive CaSR^Icc structures. In addition, the main conformation changes of CaSR during activation were also described (Liu et al., 2020). Complemented with solved crystal structures of CaSR ECD and full-length cryo-EM structures of other class C GPCR, these recent findings allow us to understand the structural framework and essential events that occur during the activation of CaSR. The overall structures of CaSR resemble the recently published structures of mGluR5 and GABA_B receptors, indicating that the structural mechanism of class C GPCRs is similar (Kim et al., 2020; Koehl et al., 2019; Mao et al., 2020; Papasergi-Scott et al., 2020; Park et al., 2020; Shaye et al., 2020).

Multiple structural and functional studies of class C GPCR have demonstrated that there are two typical conformational changes in the VFT domains during receptor activation. One is that the B–C helix angle at the interfaces of LB1–LB1 dimer sharply rotated from inactive to active state. This conformational transition of the LB1 domain is conducive to ligand binding, which is a prerequisite for receptor activation (Figure 5—figure supplement 1A,B). For example, increased glutamate affinity and occupancy in mGluR2 active conformation were observed by the mutations in B-Helix (Levitz et al., 2016). Another is that the conformation of VFT domain is converted from open to closed for the change of interdomain in one protomer, which is a landmark event during receptor activation.

We used an inhibitory nanobody to stabilize the conformation of CaSR in the inactive state. Our inactive structure shows that the B-C helix angle is about 117° and VFT domain adopts an open–open conformation (Figure 4A). The B–C helix angle has rotated approximately 28° from inactive to agonist+PAM bound state and the VFT domain rearranged from open–open configuration to closed–closed configuration during CaSR activation (Figures 4A and 5), consistent with other class C GPCR activation mechanism findings. In addition, Liu et al. developed a FRET-based conformation sensor for CaSR through fusion of SNAP-tag at its N-terminus of CaSR subunit to label with fluorophores. Their data showed that the CaSR dimer underwent a large conformational change of LB1–LB1 dimer during activation, in which the B–C helix angle rotated from inactive to active state as the fluorophores labeled the N-terminus of LB1 domain (Liu et al., 2020). We used nanobody NB-2D11 to block the proximity of LB2 domains, thus locking the CaSR in an inactivate state (Figure 6). Altogether, our structural and functional assay data suggest that our inactive cryo-EM structure represents the full inactive state of CaSR.

The rotation of LB1–LB1 domains is a watershed between inactive and intermediate states. We have reported that the crystal structure of CaSR-ECD in the open-open state (CaSR^Ioo) has the same B–C helix angle as that of the active state (CaSR^agonist+PAM), with LB1 domain configuration that is ready for L-amino acid and Ca²⁺ binding, in contrast to our cryo-EM structures of the inactive state (Figure 5—figure supplement 1A,B). Similarly, the recently reported conformation CaSR^Icc has the similar B–C helix angle as CaSR^agonist+PAM, but adopts the closed–closed VFT conformation, which is a feature of the active state. It is likely that both CaSR-ECD^Ioo and CaSR^Icc are different intermediate states during the activation of CaSR. We believe that the inhibition with NB-2D11 pushes the CaSR to a completely inactive state.

At present, the prevailing view is that the principal agonist of CaSR is extracellular Ca²⁺ (Hofer and Brown, 2003). L-amino acids, such as L-Trp, can enhance the sensitivity of CaSR toward Ca²⁺ ions (Conigrave et al., 2000), and are considered as PAMs of the receptor (Saidak et al., 2009). In line with this view, a recently reported FRET study showed that Ca²⁺ ions are sufficient to activate CaSR in the absence of L-amino acids, such that Ca²⁺ could be considered as an agonist of CaSR, whereas L-amino acids are pure PAMs of CaSR (Liu et al., 2020). However, it is interesting to note that L-amino acids or their analogs are endogenous agonists of other class C GPCRs, suggesting inconsistency from the perspective of GPCR classification and evolution.

In contrast, some studies have also shown that if Ca²⁺ concentration is higher than the threshold of 0.5 mM, L-amino acid can activate the receptor (Conigrave et al., 2004; Conigrave et al., 2000; Rey et al., 2005; Young and Rozengurt, 2002), indicating that Ca²⁺ and L-amino acid can act as co-agonists of the receptor. Using single-cell intracellular Ca²⁺ microfluorimetry, L-Trp has been shown to directly stimulate intracellular Ca²⁺ mobilization in cells stably expressing CaSR, with its efficacy and potency increase with increases in concentration of Ca²⁺ ions, hence providing direct evidence that L-amino acids are agonists of CaSR (Geng et al., 2016). However, this view has yet to be widely accepted because it is difficult to observe L-amino acids directly activating CaSR. The mode of action of Ca²⁺ ion and L-amino acids on CaSR remains controversial.

Our CaSR^agonist+PAM structure shows that TNCA and a newly identified Ca²⁺ ion bind at the interdomain cleft of the VFT module, and they both interact with both LB1 and LB2 domains to facilitate ECD closure (Figure 3A), therefore forming the closed state of the ligand-binding domain required for CaSR activation. This indicates that both TNCA and the Ca²⁺ ion contribute to the activation of CaSR. TNCA and the bound Ca²⁺ share three common binding residues S170 and D190 of LB1 domain and E297 from LB2 domain (Figure 3E,F), which suggest that the CaSR is synergistically activated by TNCA and Ca²⁺ ions.

Our experiments have shown that TNCA directly stimulate intracellular Ca²⁺ mobilization in cells stably expressing CaSR (Figure 3G), suggesting that TNCA are agonists of CaSR. The structure of CaSR shows that TNCA binds at the cleft between LB1 and LB2 domains, which is the binding site for all class C GPCR agonists (Geng et al., 2013; Geng et al., 2016; Kunishima et al., 2000; Muto et al., 2007; Tsuchiya et al., 2002), and TNCA has a binding pattern similar to that of the endogenous agonists of mGluR and GABA_B receptors (Figure 3D; Geng et al., 2016). The key coordination residues are very conserved, such as S147 and S170 (Geng et al., 2013; Geng et al., 2016; Kunishima et al., 2000; Zhang et al., 2016). Moreover, TNCA (or L-Trp) interact with residues from LB1 and LB2 to stabilize the closure of VFT, and the signal mutation of the contacting residues (T145I, S147A, S170A, Y218S, E297K) substantially reduce the function of the receptor, even if some of these residues (S147A, T145I, and Y218S) are not related to the coordinating residues of Ca²⁺, hence indicating that TNCA or L-Trp plays a key role in the activation of CaSR (Geng et al., 2016).

In addition, Ling et al. tried to determine the cryo-EM structures of CaSR in the presence of a high concentration of Ca²⁺ to address the question of whether Ca²⁺ ions alone can activate CaSR in the absence of L-Trp. Three different 3D models were obtained, in which the VFT adopted closed–closed, closed–open, and open–open conformations, and an undefined L-amino acid or its derivate was buried in the closed VFT module. However, they did not obtain the closed conformation of VFT containing only Ca²⁺ ion between the cleft (Ling et al., 2021). The results indicate that Ca²⁺ ion alone is not enough to induce the closure of the VFT module even in the presence of a high concentration of Ca²⁺ ion, and L-amino acid or its derivate is required to stabilize the closed conformation of VFT module. Altogether, L-amino acids are the endogenous agonists of CaSR, in agreement with that of other class C GPCRs.

It remains controversial whether Ca²⁺ act alone to activate CaSR in the absence of L-amino acid. Three different groups prepared CaSR samples without L-amino acids or derivatives for crystal or cryo-EM structural studies, but they all unexpectedly obtained the active structure of CaSR or CaSR ECD with closed–closed VFT conformation containing undefined ligands (Geng et al., 2016; Ling et al., 2021; Zhang et al., 2016). This ligand was subsequently identified as TNCA, which had a high affinity for CaSR, and potentiated Ca²⁺ activity (Zhang et al., 2016). As we all know, it takes a long time to purify CaSR in TNCA-free buffer for structural study; nevertheless, the endogenous TNCA would still bind to CaSR. This indicates that TNCA tightly binds to CaSR or that it is buried in closed VFT module such that it is difficult to be washed off.

Gentle washing is impossible to remove TNCA bound to CaSR in various function assays. In the absence of TNCA (or L-amino acid) and despite well-controlled assays, it is possible that endogenous TNCA would still bind to CaSR to stabilize the closed conformation of VFT. It would then appear that Ca²⁺ ions directly activate the CaSR alone as TNCA remains undetected.

In addition to the role of Ca²⁺ ion binding at the cleft of VFT module to stabilize the closed conformation of VFT, another role of the Ca²⁺ ions should be considered, in which Ca²⁺ ion is coordinated by D234, E231, and G557, and bridges the LB2 domain of one subunit and the CR domain of the second subunit. Ca²⁺ ion facilitates the formation of the active conformation of CaSR, which explain why L-amino acids (or TNCA) can activate CaSR in the presence of Ca²⁺ ion above the threshold concentration of 0.5 mM. We speculate that as endogenous TNCA exists, binds to CaSR with high affinity, and is difficult to be replaced by other added L-amino acids, it is challenging to observe whether L-amino acids can directly activate CaSR. Alternatively, the observed allosteric regulation is also a comprehensive result when we performed function assay. It needs the experience of the kinetics and dynamics of L-amino acid or TNCA binding with CaSR to confirm.

We observed that the LB2 domains approach each other during CaSR activation. Although we do not have a fully active conformation of the CaSR without agonist binding as evidence, crystal structures of a fully closed VFT modules of mGluR1 with or without agonist binding were previously reported (Kunishima et al., 2000) and have demonstrated that the proximity of both LB2 domains is an automatic process rather than an agonist-driven one. Here, we showed that NB-2D11 inhibited CaSR activation by blocking the proximity of both LB2 domains.

We analyzed how the closure of ligand bound VFT module is relayed to the signaling of 7TMDs through the CRDs. First, the rotation of LB2 domain is propagated to the large-scale transition of intersubunit 7TMDs, which leads to rearrangement of 7TMDs interface from TM5–TM6-plane/TM5–TM6-plane interface to TM6–TM6-mediated interface. Liu et al. used FRET sensor to investigate the 7TM interface rearrangement during the activation of the CaSR through a disulfide cross-linking approach. They observed a TM4–TM5-plane/TM4–TM5-plane interface in the inactive state, but a TM6–TM6 contact in the active state of the CaSR dimer (Liu et al., 2020). The conformational heterogeneity of the interface of 7TMDs in the inactive state indicates that there is a possible dynamic equilibrium between the TM4–TM5 and TM5–TM6 interfaces. TM6–TM6 contact in the active state is considered to be a hallmark of class C GPCR activation. For mGluR5 and GABA_B receptors, the 7TMDs rearrange from TM5–TM5 interface in the inactive state to TM6–TM6 interface in the active state (Kim et al., 2020; Koehl et al., 2019; Mao et al., 2020; Papasergi-Scott et al., 2020; Park et al., 2020; Shaye et al., 2020). In addition, structures of GABA_B receptor revealed some cholesterol molecules at the interface of 7TMDs (Kim et al., 2020; Mao et al., 2020; Papasergi-Scott et al., 2020; Park et al., 2020; Shaye et al., 2020). It is possible that the undefined maps between the 7TMD in our full inactive structure could be sterols that separate the dimer plane–plane interface and stabilize the inactive state (Figure 7—figure supplement 2A). In this study, we found that the conformation of ECL2 changed from the inactive to agonist+PAM state. However, the alignment of individual 7TM of both inactive and agonist+PAM states shows that the helices are well superposed, indicating that the change of ECL2 conformation is unable to drive the rearrangements of TM4 and TM5 helices and stabilize the active conformation in the TMDs (Koehl et al., 2019) This observation is consistent with findings that the active state of mGluR5 is stabilized by G proteins (Koehl et al., 2019; Manglik et al., 2015; Rosenbaum et al., 2011). It is required to determine the structure of G protein coupling CaSR to clarify the configuration of the 7TMD in the active state.

All data is available in the main text or the supplementary materials. Cryo-EM maps of active CaSR in complex with TNCA and inactive CaSR in complex with NB-2D11 have been deposited in the Electron Microscopy Data Bank under accession codes: EMD-30997 (NB-2D11 bound CaSR), EMD-30996 (TNCA bound CaSR). Atomic coordinates for the CaSR in complex with TNCA or NB-2D11 have been deposited in the Protein Data Bank under accession codes: 7E6U (NB-2D11 bound CaSR), 7E6T (TNCA bound CaSR).

1. 1. Geng Y
   2. Chen XC

   (2021) RCSB Protein Data Bank

   ID 7E6U. Cryo-EM structure of CaSR in complex with NB-2D11.

   http://www.rcsb.org/structure/unreleased/7E6U
2. 1. Chen X
   2. Wang L
   3. Ding Z
   4. Cui Q
   5. Han L
   6. Kou Y
   7. Zhang W
   8. Wang H
   9. Jia X
   10. Dai M
   11. Shi Z
   12. Li Y
   13. Li X
   14. Geng Y

   (2021) RCSB Protein Data Bank

   ID 7E6T. Cryo-EM structure of CaSR in complex with TNCA.

   http://www.rcsb.org/structure/unreleased/7E6T

1. 1. Geng Y
   2. Mosyak L
   3. Kurinov I
   4. Zuo H
   5. Sturchler E
   6. Cheng TC
   7. Subramanyam P
   8. Brown AP
   9. Brennan SC
   10. Mun H-C
   11. Bush M
   12. Chen Y
   13. Nguyen T
   14. Cao B
   15. Chang D
   16. Quick M
   17. Conigrave A
   18. Colecraft HM
   19. McDonald P
   20. Fan QR

   (2016) RCSB Protein Data Bank

   ID 5K5T. Crystal structure of the inactive form of human calcium-sensing receptor extracellular domain.

   https://www.rcsb.org/structure/5K5T
2. 1. Geng Y
   2. Mosyak L
   3. Kurinov I
   4. Zuo H
   5. Sturchler E
   6. Cheng TC
   7. Subramanyam P
   8. Brown AP
   9. Brennan SC
   10. Mun H-C
   11. Bush M
   12. Chen Y
   13. Nguyen T
   14. Cao B
   15. Chang D
   16. Quick M
   17. Conigrave A
   18. Colecraft HM
   19. McDonald P
   20. Fan QR

   (2016) RCSB Protein Data Bank

   ID 5K5S. Crystal structure of the active form of human calcium-sensing receptor extracellular domain.

   https://www.rcsb.org/structure/5K5S
3. 1. Koehl A
   2. Hu H
   3. Feng D
   4. Sun B
   5. Weis WI
   6. Skiniotis GS
   7. Mathiesen JM
   8. Kobilka BK

   (2019) RCSB Protein Data Bank

   ID 6N51. Metabotropic Glutamate Receptor 5 bound to L-quisqualate and Nb43.

   https://www.rcsb.org/structure/6N51
4. 1. Mao C
   2. Shen C
   3. Li C
   4. Shen D
   5. Xu C
   6. Zhang S
   7. Zhou R
   8. Shen Q
   9. Chen L
   10. Jiang Z
   11. Liu J
   12. Zhang Y

   (2020) RCSB Protein Data Bank

   ID 7C7Q. Cryo-EM structure of the baclofen/BHFF-bound human GABA(B) receptor in active state.

   https://www.rcsb.org/structure/7C7Q

1. 1. Adams PD
   2. Afonine PV
   3. Bunkoczi G
   4. Chen VB
   5. Davis IW
   6. Echols N
   7. Headd JJ
   8. Hung LW
   9. Kapral GJ
   10. Grosse-Kunstleve RW

   (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution

   Acta Crystallographica Section D, Biological Crystallography 66:213–221.

   https://doi.org/10.1107/S0907444909052925

   • PubMed
   • Google Scholar
2. 1. Alexander ST
   2. Hunter T
   3. Walter S
   4. Dong J
   5. Maclean D
   6. Baruch A
   7. Subramanian R
   8. Tomlinson JE

   (2015) Critical Cysteine Residues in Both the Calcium-Sensing Receptor and the Allosteric Activator AMG 416 Underlie the Mechanism of Action

   Molecular Pharmacology 88:853–865.

   https://doi.org/10.1124/mol.115.098392

   • PubMed
   • Google Scholar
3. 1. Brown EM
   2. Gamba G
   3. Riccardi D
   4. Lombardi M
   5. Butters R
   6. Kifor O
   7. Sun A
   8. Hediger MA
   9. Lytton J
   10. Hebert SC

   (1993) Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid

   Nature 366:575–580.

   https://doi.org/10.1038/366575a0

   • PubMed
   • Google Scholar
4. 1. Brown EM

   (2013) Role of the calcium-sensing receptor in extracellular calcium homeostasis

   Best Practice & Research Clinical Endocrinology & Metabolism 27:333–343.

   https://doi.org/10.1016/j.beem.2013.02.006

   • Google Scholar
5. 1. Centeno PP
   2. Herberger A
   3. Mun HC
   4. Tu C
   5. Nemeth EF
   6. Chang W
   7. Conigrave AD
   8. Ward DT

   (2019) Phosphate acts directly on the calcium-sensing receptor to stimulate parathyroid hormone secretion

   Nature Communications 10:4693.

   https://doi.org/10.1038/s41467-019-12399-9

   • PubMed
   • Google Scholar
6. 1. Chang W
   2. Tu CL
   3. Jean-Alphonse FG
   4. Herberger A
   5. Cheng Z
   6. Hwong J
   7. Ho H
   8. Li A
   9. Wang D
   10. Liu H

   (2020) PTH hypersecretion triggered by a GABAB1 and Ca(2+)-sensing receptor heterocomplex in hyperparathyroidism

   Nature Metabolism 2:243–255.

   https://doi.org/10.1038/s42255-020-0175-z

   • PubMed
   • Google Scholar
7. 1. Conigrave AD
   2. Quinn SJ
   3. Brown EM

   (2000) L-amino acid sensing by the extracellular Ca2+-sensing receptor

   PNAS 97:4814–4819.

   https://doi.org/10.1073/pnas.97.9.4814

   • PubMed
   • Google Scholar
8. 1. Conigrave AD
   2. Mun HC
   3. Delbridge L
   4. Quinn SJ
   5. Wilkinson M
   6. Brown EM

   (2004) L-amino acids regulate parathyroid hormone secretion

   The Journal of Biological Chemistry 279:38151–38159.

   https://doi.org/10.1074/jbc.M406373200

   • PubMed
   • Google Scholar
9. 1. Ellaithy A
   2. Gonzalez-Maeso J
   3. Logothetis DA
   4. Levitz J

   (2020) Structural and Biophysical Mechanisms of Class C G Protein-Coupled Receptor Function

   Trends in Biochemical Sciences 45:1049–1064.

   https://doi.org/10.1016/j.tibs.2020.07.008

   • PubMed
   • Google Scholar
10. 1. Emsley P
    2. Cowtan K

    (2004) Coot: model-building tools for molecular graphics

    Acta Crystallographica Section D, Biological Crystallography 60:2126–2132.

    https://doi.org/10.1107/S0907444904019158

    • PubMed
    • Google Scholar
11. 1. Geng Y
    2. Bush M
    3. Mosyak L
    4. Wang F
    5. Fan QR

    (2013) Structural mechanism of ligand activation in human GABA(B) receptor

    Nature 504:254–259.

    https://doi.org/10.1038/nature12725

    • PubMed
    • Google Scholar
12. 1. Geng Y
    2. Mosyak L
    3. Kurinov I
    4. Zuo H
    5. Sturchler E
    6. Cheng TC
    7. Subramanyam P
    8. Brown AP
    9. Brennan SC
    10. Mun HC

    (2016) Structural mechanism of ligand activation in human calcium-sensing receptor

    eLife 5:e13662.

    https://doi.org/10.7554/eLife.13662

    • PubMed
    • Google Scholar
13. 1. Goddard TD
    2. Huang CC
    3. Meng EC
    4. Pettersen EF
    5. Couch GS
    6. Morris JH
    7. Ferrin TE

    (2018) UCSF ChimeraX: Meeting modern challenges in visualization and analysis

    Protein Science 27:14–25.

    https://doi.org/10.1002/pro.3235

    • PubMed
    • Google Scholar
14. 1. Goehring A
    2. Lee CH
    3. Wang KH
    4. Michel JC
    5. Claxton DP
    6. Baconguis I
    7. Althoff T
    8. Fischer S
    9. Garcia KC
    10. Gouaux E

    (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    Nature Protocols 9:2574–2585.

    https://doi.org/10.1038/nprot.2014.173

    • PubMed
    • Google Scholar
15. 1. Hannan FM
    2. Kallay E
    3. Chang W
    4. Brandi ML
    5. Thakker RV

    (2018) The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases

    Nature Reviews Endocrinology 15:33–51.

    https://doi.org/10.1038/s41574-018-0115-0

    • PubMed
    • Google Scholar
16. 1. Heaney CF
    2. Kinney JW

    (2016) Role of GABA(B) receptors in learning and memory and neurological disorders

    Neuroscience and Biobehavioral Reviews 63:1–28.

    https://doi.org/10.1016/j.neubiorev.2016.01.007

    • PubMed
    • Google Scholar
17. 1. Hendy GN
    2. Guarnieri V
    3. Canaff L

    (2009) Calcium-sensing receptor and associated diseases

    Progress in Molecular Biology and Translational Science 89:31–95.

    https://doi.org/10.1016/S1877-1173(09)89003-0

    • PubMed
    • Google Scholar
18. 1. Hofer AM
    2. Brown EM

    (2003) Extracellular calcium sensing and signalling

    Nature Reviews. Molecular Cell Biology 4:530–538.

    https://doi.org/10.1038/nrm1154

    • PubMed
    • Google Scholar
19. 1. Jensen AA
    2. Brauner-Osborne H

    (2007) Allosteric modulation of the calcium-sensing receptor

    Current Neuropharmacology 5:180–186.

    https://doi.org/10.2174/157015907781695982

    • PubMed
    • Google Scholar
20. 1. Kim Y
    2. Jeong E
    3. Jeong JH
    4. Kim Y
    5. Cho Y

    (2020) Structural Basis for Activation of the Heterodimeric GABAB Receptor

    Journal of Molecular Biology 432:5966–5984.

    https://doi.org/10.1016/j.jmb.2020.09.023

    • PubMed
    • Google Scholar
21. 1. Koehl A
    2. Hu H
    3. Feng D
    4. Sun B
    5. Zhang Y
    6. Robertson MJ
    7. Chu M
    8. Kobilka TS
    9. Laeremans T
    10. Steyaert J

    (2019) Structural insights into the activation of metabotropic glutamate receptors

    Nature 566:79–84.

    https://doi.org/10.1038/s41586-019-0881-4

    • PubMed
    • Google Scholar
22. 1. Kunishima N
    2. Shimada Y
    3. Tsuji Y
    4. Sato T
    5. Yamamoto M
    6. Kumasaka T
    7. Nakanishi S
    8. Jingami H
    9. Morikawa K

    (2000) Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor

    Nature 407:971–977.

    https://doi.org/10.1038/35039564

    • PubMed
    • Google Scholar
23. 1. Leach K
    2. Gregory KJ
    3. Kufareva I
    4. Khajehali E
    5. Cook AE
    6. Abagyan R
    7. Conigrave AD
    8. Sexton PM
    9. Christopoulos A

    (2016) Towards a structural understanding of allosteric drugs at the human calcium-sensing receptor

    Cell Research 26:574–592.

    https://doi.org/10.1038/cr.2016.36

    • PubMed
    • Google Scholar
24. 1. Levitz J
    2. Habrian C
    3. Bharill S
    4. Fu Z
    5. Vafabakhsh R
    6. Isacoff EY

    (2016) Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors

    Neuron 92:143–159.

    https://doi.org/10.1016/j.neuron.2016.08.036

    • PubMed
    • Google Scholar
25. 1. Ling S
    2. Shi P
    3. Liu S
    4. Meng X
    5. Zhou Y
    6. Sun W
    7. Chang S
    8. Zhang X
    9. Zhang L
    10. Shi C

    (2021) Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca(2+) ions and L-tryptophan

    Cell Research 31:383–394.

    https://doi.org/10.1038/s41422-021-00474-0

    • PubMed
    • Google Scholar
26. 1. Liu H
    2. Yi P
    3. Zhao W
    4. Wu Y
    5. Acher F
    6. Pin JP
    7. Liu J
    8. Rondard P

    (2020) Illuminating the allosteric modulation of the calcium-sensing receptor

    PNAS 117:21711–21722.

    https://doi.org/10.1073/pnas.1922231117

    • PubMed
    • Google Scholar
27. 1. Manglik A
    2. Kim TH
    3. Masureel M
    4. Altenbach C
    5. Yang Z
    6. Hilger D
    7. Lerch MT
    8. Kobilka TS
    9. Thian FS
    10. Hubbell WL

    (2015) Structural Insights into the Dynamic Process of beta2-Adrenergic Receptor Signaling

    Cell 161:1101–1111.

    https://doi.org/10.1016/j.cell.2015.04.043

    • PubMed
    • Google Scholar
28. 1. Mao C
    2. Shen C
    3. Li C
    4. Shen DD
    5. Xu C
    6. Zhang S
    7. Zhou R
    8. Shen Q
    9. Chen LN
    10. Jiang Z

    (2020) Cryo-EM structures of inactive and active GABAB receptor

    Cell Research 30:564–573.

    https://doi.org/10.1038/s41422-020-0350-5

    • PubMed
    • Google Scholar
29. 1. Mos I
    2. Jacobsen SE
    3. Foster SR
    4. Brauner-Osborne H

    (2019) Calcium-Sensing Receptor Internalization Is beta-Arrestin-Dependent and Modulated by Allosteric Ligands

    Molecular Pharmacology 96:463–474.

    https://doi.org/10.1124/mol.119.116772

    • PubMed
    • Google Scholar
30. 1. Muto T
    2. Tsuchiya D
    3. Morikawa K
    4. Jingami H

    (2007) Structures of the extracellular regions of the group II/III metabotropic glutamate receptors

    PNAS 104:3759–3764.

    https://doi.org/10.1073/pnas.0611577104

    • PubMed
    • Google Scholar
31. 1. Nemeth EF
    2. Heaton WH
    3. Miller M
    4. Fox J
    5. Balandrin MF
    6. Van Wagenen BC
    7. Colloton M
    8. Karbon W
    9. Scherrer J
    10. Shatzen E

    (2004) Pharmacodynamics of the type II calcimimetic compound cinacalcet HCl

    The Journal of Pharmacology and Experimental Therapeutics 308:627–635.

    https://doi.org/10.1124/jpet.103.057273

    • PubMed
    • Google Scholar
32. 1. Papasergi-Scott MM
    2. Robertson MJ
    3. Seven AB
    4. Panova O
    5. Mathiesen JM
    6. Skiniotis G

    (2020) Structures of metabotropic GABAB receptor

    Nature 584:310–314.

    https://doi.org/10.1038/s41586-020-2469-4

    • PubMed
    • Google Scholar
33. 1. Pardon E
    2. Laeremans T
    3. Triest S
    4. Rasmussen SG
    5. Wohlkonig A
    6. Ruf A
    7. Muyldermans S
    8. Hol WG
    9. Kobilka BK
    10. Steyaert J

    (2014) A general protocol for the generation of Nanobodies for structural biology

    Nature Protocols 9:674–693.

    https://doi.org/10.1038/nprot.2014.039

    • PubMed
    • Google Scholar
34. 1. Park J
    2. Fu Z
    3. Frangaj A
    4. Liu J
    5. Mosyak L
    6. Shen T
    7. Slavkovich VN
    8. Ray KM
    9. Taura J
    10. Cao B

    (2020) Structure of human GABAB receptor in an inactive state

    Nature 584:304–309.

    https://doi.org/10.1038/s41586-020-2452-0

    • PubMed
    • Google Scholar
35. 1. Pin JP
    2. Bettler B

    (2016) Organization and functions of mGlu and GABAB receptor complexes

    Nature 540:60–68.

    https://doi.org/10.1038/nature20566

    • PubMed
    • Google Scholar
36. 1. Pollak MR
    2. Brown EM
    3. Chou YH
    4. Hebert SC
    5. Marx SJ
    6. Steinmann B
    7. Levi T
    8. Seidman CE
    9. Seidman JG

    (1993) Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism

    Cell 75:1297–1303.

    https://doi.org/10.1016/0092-8674(93)90617-y

    • PubMed
    • Google Scholar
37. 1. Punjani A
    2. Rubinstein JL
    3. Fleet DJ
    4. Brubaker MA

    (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

    Nature Methods 14:290–296.

    https://doi.org/10.1038/nmeth.4169

    • PubMed
    • Google Scholar
38. 1. Quinn SJ
    2. Bai M
    3. Brown EM

    (2004) pH Sensing by the calcium-sensing receptor

    The Journal of Biological Chemistry 279:37241–37249.

    https://doi.org/10.1074/jbc.M404520200

    • PubMed
    • Google Scholar
39. 1. Rey O
    2. Young SH
    3. Yuan J
    4. Slice L
    5. Rozengurt E

    (2005) Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton

    The Journal of Biological Chemistry 280:22875–22882.

    https://doi.org/10.1074/jbc.M503455200

    • PubMed
    • Google Scholar
40. 1. Rohou A
    2. Grigorieff N

    (2015) CTFFIND4: Fast and accurate defocus estimation from electron micrographs

    Journal of Structural Biology 192:216–221.

    https://doi.org/10.1016/j.jsb.2015.08.008

    • PubMed
    • Google Scholar
41. 1. Rosenbaum DM
    2. Zhang C
    3. Lyons JA
    4. Holl R
    5. Aragao D
    6. Arlow DH
    7. Rasmussen SG
    8. Choi HJ
    9. Devree BT
    10. Sunahara RK

    (2011) Structure and function of an irreversible agonist-beta(2) adrenoceptor complex

    Nature 469:236–240.

    https://doi.org/10.1038/nature09665

    • PubMed
    • Google Scholar
42. 1. Saidak Z
    2. Boudot C
    3. Abdoune R
    4. Petit L
    5. Brazier M
    6. Mentaverri R
    7. Kamel S

    (2009) Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

    Experimental Cell Research 315:2072–2080.

    https://doi.org/10.1016/j.yexcr.2009.03.003

    • PubMed
    • Google Scholar
43. 1. Shaye H
    2. Ishchenko A
    3. Lam JH
    4. Han GW
    5. Xue L
    6. Rondard P
    7. Pin JP
    8. Katritch V
    9. Gati C
    10. Cherezov V

    (2020) Structural basis of the activation of a metabotropic GABA receptor

    Nature 584:298–303.

    https://doi.org/10.1038/s41586-020-2408-4

    • PubMed
    • Google Scholar
44. 1. Tsuchiya D
    2. Kunishima N
    3. Kamiya N
    4. Jingami H
    5. Morikawa K

    (2002) Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+

    PNAS 99:2660–2665.

    https://doi.org/10.1073/pnas.052708599

    • PubMed
    • Google Scholar
45. 1. Walter S
    2. Baruch A
    3. Dong J
    4. Tomlinson JE
    5. Alexander ST
    6. Janes J
    7. Hunter T
    8. Yin Q
    9. Maclean D
    10. Bell G

    (2013) Pharmacology of AMG 416 (Velcalcetide), a novel peptide agonist of the calcium-sensing receptor, for the treatment of secondary hyperparathyroidism in hemodialysis patients

    The Journal of Pharmacology and Experimental Therapeutics 346:229–240.

    https://doi.org/10.1124/jpet.113.204834

    • PubMed
    • Google Scholar
46. 1. Ward BK
    2. Magno AL
    3. Walsh JP
    4. Ratajczak T

    (2012) The role of the calcium-sensing receptor in human disease

    Clinical Biochemistry 45:943–953.

    https://doi.org/10.1016/j.clinbiochem.2012.03.034

    • PubMed
    • Google Scholar
47. 1. Xue L
    2. Rovira X
    3. Scholler P
    4. Zhao H
    5. Liu J
    6. Pin JP
    7. Rondard P

    (2015) Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer

    Nature Chemical Biology 11:134–140.

    https://doi.org/10.1038/nchembio.1711

    • PubMed
    • Google Scholar
48. 1. Young SH
    2. Rozengurt E

    (2002) Amino acids and ca2+ stimulate different patterns of ca2+ oscillations through the ca2+-sensing receptor

    American Journal of Physiology. Cell Physiology 282:C1414–C1422.

    https://doi.org/10.1152/ajpcell.00432.2001

    • PubMed
    • Google Scholar
49. 1. Zhang C
    2. Zhang T
    3. Zou J
    4. Miller CL
    5. Gorkhali R
    6. Yang JY
    7. Schilmiller A
    8. Wang S
    9. Huang K
    10. Brown EM

    (2016) Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist

    Science Advances 2:e1600241.

    https://doi.org/10.1126/sciadv.1600241

    • PubMed
    • Google Scholar
50. 1. Zheng SQ
    2. Palovcak E
    3. Armache JP
    4. Verba KA
    5. Cheng Y
    6. Agard DA

    (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

    Nature Methods 14:331–332.

    https://doi.org/10.1038/nmeth.4193

    • PubMed
    • Google Scholar
51. 1. Zivanov J
    2. Nakane T
    3. Scheres SHW

    (2020) Estimation of high-order aberrations and anisotropic magnification from Cryo-em data sets in relion-3.1

    IUCrJ 7:253–267.

    https://doi.org/10.1107/s2052252520000081

    • PubMed
    • Google Scholar

Author details

1. Xiaochen Chen

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Lu Wang, Qianqian Cui and Zhanyu Ding

   Competing interests

   None

   "This ORCID iD identifies the author of this article:" 0000-0002-0426-9547

2. Lu Wang

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Data curation, Investigation, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Xiaochen Chen, Qianqian Cui and Zhanyu Ding

   Competing interests

   None

   "This ORCID iD identifies the author of this article:" 0000-0001-6336-5806

3. Qianqian Cui

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation, Writing – review and editing

   Contributed equally with

   Xiaochen Chen, Lu Wang and Zhanyu Ding

   Competing interests

   none

   "This ORCID iD identifies the author of this article:" 0000-0002-5962-9298

4. Zhanyu Ding

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Data curation, Investigation, Validation, Writing – original draft

   Contributed equally with

   Xiaochen Chen, Lu Wang and Qianqian Cui

   Competing interests

   None

   "This ORCID iD identifies the author of this article:" 0000-0002-8136-2243

5. Li Han

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   None

6. Yongjun Kou

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   None

7. Wenqing Zhang

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   None

8. Haonan Wang

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   None

9. Xiaomin Jia

   The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation

   Competing interests

   None

10. Mei Dai

    The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Contribution

    Investigation

    Competing interests

    None

11. Zhenzhong Shi

    The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Contribution

    Investigation

    Competing interests

    None

12. Yuying Li

    The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Contribution

    Investigation

    Competing interests

    None

13. Xiyang Li

    The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Contribution

    Investigation

    Competing interests

    None

14. Yong Geng

    1. The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
    2. University of Chinese Academy of Sciences, Beijing, China

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft

    For correspondence

    gengyong@simm.ac.cn

    Competing interests

    None

    "This ORCID iD identifies the author of this article:" 0000-0001-7144-3878

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