Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor
Jianxin Hu, Guadalupe Reyes-Cruz, Paul K. Goldsmith, Nicole M. Gantt, Jeffery L. Miller, Allen M. Spiegel
We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. Introduction: The human Ca2+ receptor (CaR), which plays a central role in the regulation of [Ca2+]0 homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. Materials and Methods: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca2+ stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca2+ and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. Results: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca2+ was also increased by binding of 5C8 and decreased by binding of 1A8. Conclusions: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.
Journal of Bone and Mineral Research
https://doi.org/10.1359/jbmr.070111
Chimeric receptor
Extracellular domain
Human Ca receptor
Venus flytrap-like domain
10.1359/jbmr.070111
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Droseraceae Medicine & Life Sciences
Monoclonal Antibodies Medicine & Life Sciences
Takifugu Medicine & Life Sciences
Epitopes Medicine & Life Sciences
Flow Cytometry Medicine & Life Sciences
Hyperparathyroidism Medicine & Life Sciences
HEK293 Cells Medicine & Life Sciences
Hu, J., Reyes-Cruz, G., Goldsmith, P. K., Gantt, N. M., Miller, J. L., & Spiegel, A. M. (2007). Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor. Journal of Bone and Mineral Research, 22(4), 601-608. https://doi.org/10.1359/jbmr.070111
Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor. / Hu, Jianxin; Reyes-Cruz, Guadalupe; Goldsmith, Paul K.; Gantt, Nicole M.; Miller, Jeffery L.; Spiegel, Allen M.
In: Journal of Bone and Mineral Research, Vol. 22, No. 4, 04.2007, p. 601-608.
Hu, J, Reyes-Cruz, G, Goldsmith, PK, Gantt, NM, Miller, JL & Spiegel, AM 2007, 'Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor', Journal of Bone and Mineral Research, vol. 22, no. 4, pp. 601-608. https://doi.org/10.1359/jbmr.070111
Hu J, Reyes-Cruz G, Goldsmith PK, Gantt NM, Miller JL, Spiegel AM. Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor. Journal of Bone and Mineral Research. 2007 Apr;22(4):601-608. https://doi.org/10.1359/jbmr.070111
Hu, Jianxin ; Reyes-Cruz, Guadalupe ; Goldsmith, Paul K. ; Gantt, Nicole M. ; Miller, Jeffery L. ; Spiegel, Allen M. / Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor. In: Journal of Bone and Mineral Research. 2007 ; Vol. 22, No. 4. pp. 601-608.
@article{ef1059be5a4347a8837e068522ed41cf,
title = "Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor",
abstract = "We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. Introduction: The human Ca2+ receptor (CaR), which plays a central role in the regulation of [Ca2+]0 homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. Materials and Methods: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca2+ stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca2+ and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. Results: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca2+ was also increased by binding of 5C8 and decreased by binding of 1A8. Conclusions: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.",
keywords = "Chimeric receptor, Extracellular domain, Human Ca receptor, Monoclonal antibodies, Venus flytrap-like domain",
author = "Jianxin Hu and Guadalupe Reyes-Cruz and Goldsmith, {Paul K.} and Gantt, {Nicole M.} and Miller, {Jeffery L.} and Spiegel, {Allen M.}",
doi = "10.1359/jbmr.070111",
journal = "Journal of Bone and Mineral Research",
T1 - Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor
AU - Hu, Jianxin
AU - Reyes-Cruz, Guadalupe
AU - Goldsmith, Paul K.
AU - Gantt, Nicole M.
AU - Miller, Jeffery L.
AU - Spiegel, Allen M.
N2 - We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. Introduction: The human Ca2+ receptor (CaR), which plays a central role in the regulation of [Ca2+]0 homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. Materials and Methods: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca2+ stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca2+ and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. Results: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca2+ was also increased by binding of 5C8 and decreased by binding of 1A8. Conclusions: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.
AB - We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. Introduction: The human Ca2+ receptor (CaR), which plays a central role in the regulation of [Ca2+]0 homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. Materials and Methods: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca2+ stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca2+ and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. Results: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca2+ was also increased by binding of 5C8 and decreased by binding of 1A8. Conclusions: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.
KW - Chimeric receptor
KW - Extracellular domain
KW - Human Ca receptor
KW - Monoclonal antibodies
KW - Venus flytrap-like domain
U2 - 10.1359/jbmr.070111
DO - 10.1359/jbmr.070111
JO - Journal of Bone and Mineral Research
JF - Journal of Bone and Mineral Research