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Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor
Chen Zhang1, Nagaraju Mulpuri2, Fadil M Hannan3
1Department of Chemistry, Georgia State University, Atlanta, Georgia, United States of America; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, United States of America.
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.
Insights
Mutations in the Ca(2+)-sensing receptor (CaSR) can cause calcium disorders. This study reveals how specific mutations affect CaSR cooperativity, impacting calcium homeostasis and offering insights into potential therapeutic strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Computational Biology
Background:
- The Ca(2+)-sensing receptor (CaSR) is crucial for maintaining calcium homeostasis by sensing extracellular Ca(2+) levels.
- Mutations in the CaSR, particularly in the Venus flytrap domain (VFTD), are linked to hypercalcemic and hypocalcemic disorders.
- Understanding the functional impact of these mutations on CaSR cooperativity is essential for deciphering disease mechanisms.
Purpose of the Study:
- To investigate the functional consequences of specific CaSR mutations (L173P, P221Q, L173F, P221L) on receptor cooperativity.
- To explore the role of the amino acid L-phenylalanine (L-Phe) in modulating CaSR activity and cooperativity.
- To elucidate the structural dynamics underlying loss-of-function and gain-of-function CaSR mutations using computational simulations.
Main Methods:
- Functional assays measuring Ca(2+)-induced intracellular Ca(2+) oscillations, inositol-1-phosphate (IP1) accumulation, and ERK1/2 activity.
- Computational simulations to analyze the dynamics and correlated motions within VFTD mutants.
- In silico and in vitro experiments to assess the effect of L-Phe on wild-type (WT) and mutant CaSR activity.
Main Results:
- Loss-of-function mutations (L173P, P221Q) impaired CaSR positive cooperativity, while gain-of-function mutations (L173F, P221L) enhanced it.
- Computational simulations revealed disrupted correlated motions in loss-of-function mutants and enhanced motions in gain-of-function mutants.
- L-Phe rescued the response of the P221Q mutant, demonstrating its ability to enhance CaSR positive cooperativity.
Conclusions:
- Residues Leu(173) and Pro(221) are critical for CaSR's homo- and heterotropic cooperative regulation.
- The VFTD hinge region plays a key role in modulating CaSR cooperativity, influencing calcium homeostasis.
- Findings provide insights into CaSR regulation and potential therapeutic targets for calcium-related disorders.
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