Abstract
The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.
Highlights
The G protein-coupled Ca2؉-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca2؉ and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca2؉ concentration ([Ca2؉]i)
To determine whether [Ca2ϩ]o and aromatic amino acids differ in their ability to trigger the synthesis of these second messengers via the CaR, we used a set of biosensors to monitor, in real time and in the same cells, the synthesis of Ins(1,4,5)P3 and DAG simultaneously with [Ca2ϩ]i oscillations
Phenylalanine or [Ca2ϩ]e Stimulation of the CaR—The results presented in Figs. 1 and 2, and our previous studies (9, 11), support the notion that [Ca2ϩ]o-elicited CaR activation leads to stimulation of the Gq/phospholipase C (PLC) pathway leading to the production of sinusoidal [Ca2ϩ]i oscillations
Summary
CaR, Ca2ϩ-sensing receptor; [Ca2ϩ]o, extracellular Ca2ϩ; [Ca2ϩ]i, intracellular Ca2ϩ; GPCR, G protein-coupled receptor; PKC, protein kinase C; PKD, protein kinase D; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; HEK, human embryonic kidney; MEF, murine embryonic fibroblasts; RKϪ/Ϫ, rhodopsin kinase knock-out mice; RPTEC, renal proximal tubule epithelial cells; RFP, red fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; PHD, pleckstrin homology domain; PLC, phospholipase C; DAG, diacylglycerol. Aromatic amino acid stimulation of the CaR induces repetitive, low frequency [Ca2ϩ]i spikes that return to the base-line level, a pattern known as transient oscillations. The transient [Ca2ϩ]i oscillations produced by the CaR in response to amino acid stimulation appear to be mediated by a different pathway, but the mechanism(s) involved remained poorly understood. We examined whether sinusoidal and transient [Ca2ϩ]i oscillations produced by the CaR in response to Ca2ϩ or L-phenylalanine are mediated by different pathways. We identified a novel pathway that mediates transient [Ca2ϩ]i oscillations produced by the CaR in response to amino acids, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the. Our model envisages that Ca2ϩ or amino acids induce distinct conformational states of the CaR, providing for the possibility of differential coupling to downstream signaling pathways
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