Intracellular Ca²⁺oscillations generated via the Ca²⁺-sensing receptor are mediated by negative feedback by PKCα at Thr⁸⁸⁸

Abstract

To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 μM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).

Inhibitors of DAG metabolism suppress CCR2 signalling in human monocytes

“…Due to the diverse repertoire of PKC isoforms expressed, we employed pharmacological tools that allowed discrimination between classical DAG and Ca 2+ ‐dependent isoforms (α, β, and γ; blocked by Gö6976) and DAG dependent but Ca 2+ ‐independent isoforms (δ, ε, η, or θ; blocked by Gö6983); Gö6983 also blocks α, β, and γ isoforms (Goekjian & Jirousek, ; Gschwendt et al, ). Gö6983 100‐nM (Young, Rey, Sinnett‐Smith, & Rozengurt, ) had no effect on the control Ca 2+ response to CCL2 (Figure a); however, 100‐nM Gö6976 (Lin, Leu, Huang, & Tsai, ) significantly inhibited the response (Figure a). Gö6983 could rescue the response inhibited by OAG (Figure b), R59949 (Figure c), or RHC80267 (Figure d), though Gö6976 could not (Figure b–d).…”

Intracellular Ca 2+ oscillations generated via the extracellular Ca 2+ -sensing receptor (CaSR) in response to extracellular Ca 2+ or l -phenylalanine: Impact of the highly conservative mutation Ser170Thr

“…Human Embryonic Kidney (HEK-293) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified incubator under 10% CO 2 and 90% air at 37°C, as described previously [13,25,32]. For experimental purposes, cells were plated onto 18-mm diameter glass coverslips inside 35-mm plastic dishes, where they could be dually transiently transfected with a plasmid encoding the human CaSR or a mutant CaSR receptor (CaSR S170T ).…”

“…Oscillatory changes in [Ca 2+ ] i in response to receptor stimulation is a fundamental mechanism of cell signaling implicated in the regulation of Ca 2+ - and calmodulin-dependent protein kinase II [19], conventional protein kinase C (PKC) isoforms [15,20], mitochondrial function [21,22], and nuclear transcriptional activity leading to differential gene expression [23,24]. We proposed that [Ca 2+ ] i oscillations induced by activation of the CaSR in response to an increase in extracellular Ca 2+ results from negative feedback involving PKC-mediated phosphorylation of the CaSR at the inhibitory residue Thr 888 [14,25]. In addition to its role as sensor of [Ca 2+ ] e , the CaSR is also stimulated by aromatic amino acids [26] which, like [Ca 2+ ] e , induce striking and lasting CaSR-mediated [Ca 2+ ] i oscillations [13,15,16].…”

The Calcium-Sensing Receptor and the Parathyroid: Past, Present, Future

“…The CaSR mediates, for example, the activation of various protein kinases including protein kinase C isoforms, which negatively modulate CaSR function (Jiang et al, 2002; Davies et al, 2007; Lazarus et al, 2011; Young et al, 2014), and the mitogen activated protein (MAP) kinases ERK 1/2 , p38 and JNK (Kifor et al, 2001; Tfelt-Hansen et al, 2003; review: Conigrave and Ward, 2013). The roles of protein kinases in CaSR-mediated inhibitory control of PTH secretion are not well-understood but ERK 1/2 appears to contribute (Corbetta et al, 2002) and could be activated downstream of either G q/11 or G i (review: Conigrave and Ward, 2013).…”