Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.

Chen Zhang, Nagaraju Mulpuri, Fadil M Hannan, M. Andrew Nesbit, Rajesh V Thakker, Donald Hamelberg, Edward M Brown, Jenny J Yang

Research output: Contribution to journalArticle

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Abstract

The Ca(2+)-sensing receptor (CaSR) regulates Ca(2+) homeostasis in the body by monitoring extracellular levels of Ca(2+) ([Ca(2+)]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+)]o-induced [Ca(2+)]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+)]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+)]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173), which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221) and Leu(173) are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.
LanguageEnglish
Pagese113622
JournalPLoS ONE
Volume9
Issue number11
Publication statusPublished - 2014

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Droseraceae
Calcium-Sensing Receptors
mutation
calcium
mutants
Dionaea muscipula
Mutation
receptors
Mitogen-Activated Protein Kinase 1
Extracellular Signal-Regulated MAP Kinases
Hinges
Computer Simulation
Homeostasis
Chemical activation
Binding Sites
cooperatives
Amino Acids
Monitoring
Homo
mitogen-activated protein kinase

Keywords

  • Calcium sensing receptor

Cite this

Zhang, C., Mulpuri, N., Hannan, F. M., Nesbit, M. A., Thakker, R. V., Hamelberg, D., ... Yang, J. J. (2014). Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations. PLoS ONE, 9(11), e113622.
Zhang, Chen ; Mulpuri, Nagaraju ; Hannan, Fadil M ; Nesbit, M. Andrew ; Thakker, Rajesh V ; Hamelberg, Donald ; Brown, Edward M ; Yang, Jenny J. / Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations. In: PLoS ONE. 2014 ; Vol. 9, No. 11. pp. e113622.
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abstract = "The Ca(2+)-sensing receptor (CaSR) regulates Ca(2+) homeostasis in the body by monitoring extracellular levels of Ca(2+) ([Ca(2+)]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+)]o-induced [Ca(2+)]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+)]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+)]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173), which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221) and Leu(173) are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.",
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Zhang, C, Mulpuri, N, Hannan, FM, Nesbit, MA, Thakker, RV, Hamelberg, D, Brown, EM & Yang, JJ 2014, 'Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.', PLoS ONE, vol. 9, no. 11, pp. e113622.

Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations. / Zhang, Chen; Mulpuri, Nagaraju; Hannan, Fadil M; Nesbit, M. Andrew; Thakker, Rajesh V; Hamelberg, Donald; Brown, Edward M; Yang, Jenny J.

In: PLoS ONE, Vol. 9, No. 11, 2014, p. e113622.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.

AU - Zhang, Chen

AU - Mulpuri, Nagaraju

AU - Hannan, Fadil M

AU - Nesbit, M. Andrew

AU - Thakker, Rajesh V

AU - Hamelberg, Donald

AU - Brown, Edward M

AU - Yang, Jenny J

PY - 2014

Y1 - 2014

N2 - The Ca(2+)-sensing receptor (CaSR) regulates Ca(2+) homeostasis in the body by monitoring extracellular levels of Ca(2+) ([Ca(2+)]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+)]o-induced [Ca(2+)]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+)]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+)]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173), which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221) and Leu(173) are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.

AB - The Ca(2+)-sensing receptor (CaSR) regulates Ca(2+) homeostasis in the body by monitoring extracellular levels of Ca(2+) ([Ca(2+)]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+)]o-induced [Ca(2+)]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+)]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+)]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173), which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221) and Leu(173) are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.

KW - Calcium sensing receptor

M3 - Article

VL - 9

SP - e113622

JO - PLoS ONE

T2 - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 11

ER -