Abstract: The calcium-sensing receptor (CaSR) plays a central role in maintaining systemic calcium homeostasis. In the treatment of secondary hyperparathyroidism (SHPT) associated with chronic kidney disease (CKD), CaSR is targeted by calcimimetic drugs. Recent cryo-electron microscopy (cryo-EM) studies have begun to reveal how CaSR is modulated by various allosteric modulators and signals through G proteins. However, the mechanisms of CaSR activation by the novel calcimimetic (upacicalcet) and the aminoglycoside antibiotic (neomycin) remain elusive. In this study, we present high-resolution cryo-EM structures of the human CaSR homodimer complexed with either upacicalcet or neomycin. Coupled with functional studies, our results reveal that both drugs target the extracellular region of the receptor. Upacicalcet occupies the amino acid-binding site of the Venus flytrap (VFT) domain, promoting VFT closure, whereas neomycin occupies a calcium-binding site at the homodimer interface at the base of the VFT, thus inducing the proximity of the two protomers. These findings reveal distinct molecular mechanisms of CaSR modulation by drug molecules, highlight the diversity of ligand-dependent regulatory modes, and provide a structural foundation for the rational design of future CaSR-targeting therapeutics.

细胞外调控分子激活钙敏感受体的结构基础

[1]Actkins KV, Beasley HK, Faucon AB, et al., 2021. Calcium-sensing receptor polymorphisms at rs1801725 are associated with increased risk of secondary malignancies. J Pers Med, 11(7):642.

[2]Asensio JL, Hidalgo A, Cuesta I, et al., 2002. Experimental evidence for the existence of non-exo-anomeric conformations in branched oligosaccharides: NMR analysis of the structure and dynamics of aminoglycosides of the neomycin family. Chemistry, 8(22):5228-5240. https://doi.org/10.1002/1521-3765(20021115)8:22<5228::AID-CHEM5228>3.0.CO;2-L

[3]Chang WH, Tu CL, Jean-Alphonse FG, et al., 2020. PTH hypersecretion triggered by a GABA_B1 and Ca²⁺-sensing receptor heterocomplex in hyperparathyroidism. Nat Metab, 2(3):243-255.

[4]Chattopadhyay N, 2006. Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy. Am J Physiol Endocrinol Metab, 290(5):E761-E770.

[5]Chen LN, Zhou H, Xi K, et al., 2025. Proton perception and activation of a proton-sensing GPCR. Mol Cell, 85(8):1640-1657.e8.

[6]Chen VB, Arendall WB III, Headd JJ, et al., 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 66(Pt 1):12-21.

[7]Chen XC, Wang L, Cui QQ, et al., 2021. Structural insights into the activation of human calcium-sensing receptor. eLife, 10:e68578.

[8]Cianferotti L, Gomes AR, Fabbri S, et al., 2015. The calcium-sensing receptor in bone metabolism: from bench to bedside and back. Osteoporos Int, 26(8):2055-2071.

[9]Diao JY, DeBono A, Josephs TM, et al., 2021. Therapeutic opportunities of targeting allosteric binding sites on the calcium-sensing receptor. ACS Pharmacol Transl Sci, 4(2):666-679.

[10]Durocher Y, Perret S, Kamen A, 2001. Recombinant protein production by transient transfection of suspension-growing cells. In: Merten OW, Mattanovich D, Lang C, et al. (Eds.), Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology: Selected Articles from the Meeting of the EFB Section on Microbial Physiology. Springer, Dordrecht, p.331-337.

[11]Emsley P, Lohkamp B, Scott WG, et al., 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr, 66(Pt 4):486-501.

[12]Gao Y, Robertson MJ, Rahman SN, et al., 2021. Asymmetric activation of the calcium-sensing receptor homodimer. Nature, 595(7867):455-459.

[13]Geng Y, Mosyak L, Kurinov I, et al., 2016. Structural mechanism of ligand activation in human calcium-sensing receptor. eLife, 5:e13662.

[14]Gerace E, Moazed D, 2015. Affinity pull-down of proteins using anti-FLAG M2 agarose beads. Methods Enzymol, 559:99-110.

[15]Goralski T, Ram JL, 2022. Extracellular calcium receptor as a target for glutathione and its derivatives. Int J Mol Sci, 23(2):717.

[16]Gorvin CM, 2023. Recent advances in calcium-sensing receptor structures and signaling pathways. Prog Mol Biol Transl Sci, 195:121-135.

[17]Goto M, Nishimura G, Sato H, et al., 2023. Pharmacological profile of upacicalcet, a novel positive allosteric modulator of calcium-sensing receptor, in vitro and in vivo. Eur J Pharmacol, 956:175936.

[18]Grant MP, Stepanchick A, Breitwieser GE, 2012. Calcium signaling regulates trafficking of familial hypocalciuric hypercalcemia (FHH) mutants of the calcium sensing receptor. Mol Endocrinol, 26(12):2081-2091.

[19]Hamano N, Komaba H, Fukagawa M, 2017. Etelcalcetide for the treatment of secondary hyperparathyroidism. Expert Opin Pharmacother, 18(5):529-534.

[20]Hamano T, Koiwa F, Isaka Y, et al., 2025. Long-term efficacy and safety of upacicalcet in Japanese hemodialysis patients with secondary hyperparathyroidism: open-label 52-week study. Am J Nephrol, 56(1):70-84.

[21]Hannan FM, Nesbit MA, Zhang C, et al., 2012. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites. Hum Mol Genet, 21(12):2768-2778.

[22]Hannan FM, Kallay E, Chang WH, et al., 2019. The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol, 15:33-51.

[23]He F, Wu CG, Gao Y, et al., 2024. Allosteric modulation and G-protein selectivity of the Ca²⁺-sensing receptor. Nature, 626(8001):1141-1148.

[24]Hofer AM, Brown EM, 2003. Extracellular calcium sensing and signalling. Nat Rev Mol Cell Biol, 4(7):530-538.

[25]Liebschner D, Afonine PV, Baker ML, et al., 2019. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol, 75(Pt 10):861-877.

[26]Ling SL, Shi P, Liu SL, et al., 2021. Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca²⁺ ions and L-tryptophan. Cell Res, 31(4):383-394.

[27]Liu FY, Wu CG, Tu CL, et al., 2024. Large library docking identifies positive allosteric modulators of the calcium-sensing receptor. Science, 385(6715):eado1868.

[28]Liu GM, Cao W, Jia G, et al., 2018. Calcium-sensing receptor in nutrient sensing: an insight into the modulation of intestinal homoeostasis. Br J Nutr, 120(8):881-890.

[29]McLarnon SJ, Holden D, Ward DT, et al., 2002. Aminoglycoside antibiotics induce pH-sensitive activation of the calcium-sensing receptor. Biochem Biophys Res Commun, 297(1):71-77.

[30]Morales-Perez CL, Noviello CM, Hibbs RE, 2016. Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure, 24(5):797-805.

[31]Moriarty NW, Grosse-Kunstleve RW, Adams PD, 2009. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr D Biol Crystallogr, 65(Pt 10):1074-1080.

[32]Nemeth EF, van Wagenen BC, Balandrin MF, 2018. Discovery and development of calcimimetic and calcilytic compounds. Prog Med Chem, 57:1-86.

[33]Olsen RHJ, DiBerto JF, English JG, et al., 2020. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat Chem Biol, 16(8):841-849.

[34]Owen JL, Cheng SX, Ge Y, et al., 2016. The role of the calcium-sensing receptor in gastrointestinal inflammation. Semin Cell Dev Biol, 49:44-51.

[35]Patel J, Bridgeman MB, 2018. Etelcalcetide (Parsabiv) for secondary hyperparathyroidism in adults with chronic kidney disease on hemodialysis. Pharm Ther, 43(7):396-399.

[36]Pathalys Pharma, 2025a. A phase 3 randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of dose-titrated PLS240 in the treatment of secondary hyperparathyroidism in individuals with end stage kidney disease on hemodialysis (PATH-1) with an open-label extension (identifier: NCT05832931). ClinicalTrials.gov. https://clinicaltrials.gov/study/NCT05832931

[37]Pathalys Pharma, 2025b. A phase 3 randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of dose-titrated PLS240 in the treatment of secondary hyperparathyroidism in individuals with end stage kidney disease on hemodialysis (PATH-2) with an open-label extension (identifier: NCT05836220). ClinicalTrials.gov. https://clinicaltrials.gov/study/NCT05836220

[38]Pettersen EF, Goddard TD, Huang CC, et al., 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem, 25(13):1605-1612.

[39]Pettersen EF, Goddard TD, Huang CC, et al., 2021. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci, 30(1):70-82.

[40]Punjani A, Rubinstein JL, Fleet DJ, et al., 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods, 14(3):290-296.

[41]Riccardi D, Brown EM, 2010. Physiology and pathophysiology of the calcium-sensing receptor in the kidney. Am J Physiol Renal Physiol, 298(3):F485-F499.

[42]Ruat M, Traiffort E, 2013. Roles of the calcium sensing receptor in the central nervous system. Best Pract Res Clin Endocrinol Metab, 27(3):429-442.

[43]Schepelmann M, Yarova PL, Lopez-Fernandez I, et al., 2016. The vascular Ca²⁺-sensing receptor regulates blood vessel tone and blood pressure. Am J Physiol Cell Physiol, 310(3):C193-C204.

[44]Shen CS, Mao CY, Xu CJ, et al., 2021. Structural basis of GABA_B receptor–G_i protein coupling. Nature, 594(7864):594-598.

[45]Shigematsu T, Koiwa F, Isaka Y, et al., 2023. Efficacy and safety of upacicalcet in hemodialysis patients with secondary hyperparathyroidism: a randomized placebo-controlled trial. Clin J Am Soc Nephrol, 18(10):1300-1309.

[46]Squires PE, Harris TE, Persaud SJ, et al., 2000. The extracellular calcium-sensing receptor on human beta-cells negatively modulates insulin secretion. Diabetes, 49(3):409-417.

[47]Squires PE, Jones PM, Younis MYG, et al., 2014. The calcium-sensing receptor and β-cell function. Vitam Horm, 95:249-267.

[48]Tian L, Andrews C, Yan QY, et al., 2024. Molecular regulation of calcium-sensing receptor (CaSR)-mediated signaling. Chronic Dis Transl Med, 10(3):167-194.

[49]Tsai SH, Kan WC, Jhen RN, et al., 2024. Secondary hyperparathyroidism in chronic kidney disease: a narrative review focus on therapeutic strategy. Clin Med (Lond), 24(5):100238.

[50]Ward DT, McLarnon SJ, Riccardi D, 2002. Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor. J Am Soc Nephrol, 13(6):1481-1489.

[51]Wen TL, Wang ZY, Chen XZ, et al., 2021. Structural basis for activation and allosteric modulation of full-length calcium-sensing receptor. Sci Adv, 7(23):eabg1483.

[52]Xu PY, Huang SJ, Krumm BE, et al., 2023. Structural genomics of the human dopamine receptor system. Cell Res, 33(8):604-616.

[53]Yamaguchi H, Kitajima S, Suzuki H, et al., 2025. Cryo-EM structure of the calcium-sensing receptor complexed with the kokumi substance γ-glutamyl-valyl-glycine. Sci Rep, 15:3894.

[54]Zhang C, Zhang T, Zou J, et al., 2016. Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist. Sci Adv, 2(5):e1600241.

[55]Zuo H, Park J, Frangaj A, et al., 2024. Promiscuous G-protein activation by the calcium-sensing receptor. Nature, 629(8011):481-488.

Similar articles