Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation - PubMed (original) (raw)

Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation

Heather J Lee et al. Biochem J. 2007.

Abstract

The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.

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Figures

Figure 1

Figure 1. Activation of Ca2+o-stimulated ERK1/2 by calcimimetic NPS R467 in CaR-expressing HEK-293 cells

HEK-293 cells that stably expressed the human wild-type CaR were incubated in PSS that contained various Ca2+o concentrations in the absence or presence of 1 μM NPS R467. The incubations were stopped by the addition of ice-cold PBS and the cells were lysed. In (A) and (B), the samples were processed by SDS/PAGE and Western blotting, and the blots were developed for total and phosphorylated ERK1/2 and then quantified as described in the Experimental section. The data were then expressed as a percentage of the maximum level of control phosphorylation for each experiment. (A) A representative Western blot for total (upper panel) and phosphorylated (lower panel) ERK1/2. Molecular masses are indicated in kDa. (B) Ca2+o concentration–response data showing the effect of 1 μM NPS R467 (R-467) on Ca2+o-stimulated ERK1/2 phosphorylation detected by Western blotting. In (C), the samples were processed for SureFire cellular ERK1/2 analysis as described in the Experimental section. ○, Control; ▲, NPS R467.

Figure 2

Figure 2. Effects of L-phenylalanine on Ca2+o-stimulated ERK1/2 phosphorylation in CaR-expressing HEK-293 cells

CaR-expressing HEK-293 cells were incubated in the presence of 2 mM Ca2+ in the absence (−) or presence (+) of 10 mM

L

-phenylalanine for 10 min. The incubations were then stopped by exposure to ice-cold PBS, and the cells were lysed and processed by SDS/PAGE and Western blotting. Films were developed by ECL® as described in the Experimental section.

Figure 3

Figure 3. Effects of L-phenylalanine on the Ca2+o-sensitivity of ERK1/2 phosphorylation in CaR-expressing HEK-293 cells

Wild-type CaR-expressing HEK-293 cells were incubated (A and B) in the presence of various Ca2+ concentrations (0.5–6.0 mM) in the absence or presence of 10 mM

L

-phenylalanine for 10 min. The incubations were stopped by exposure to ice-cold PBS, and the cells were then lysed and processed by SDS/PAGE and Western blotting as described in the Experimental section. All Ca2+o-concentration-dependent data were expressed as a percentage of the maximum level of phosphorylation obtained for each control data set. (A) Representative blot demonstrating a small enhancement of the Ca2+-sensitivity of ERK1/2 phosphorylation by 10 mM

L

-phenylalanine in the region of the EC50 for Ca2+o (approx. 2.0 mM). (B) Ca2+-concentration-dependent activation of ERK1/2 phosphorylation in the absence or presence of 10 mM

L

-phenylalanine from a total of 25 experiments. (C) CaR-expressing HEK-293 cells were incubated in the presence of various Ca2+o concentrations between 0.5 and 10 mM and then processed for the SureFire ERK1/2 assay as described in the Experimental section (_n_=14). ○, Control; ▲, 10 mM

L

-phenylalanine.

Figure 4

Figure 4. Concentration-dependence and stereoselectivity of L-phenylalanine and effects of various other amino acids

HEK-293 cells that stably expressed the wild-type CaR were pre-incubated for 30 min and then exposed to (A) various concentrations of Ca2+ (0.5, 2.0 and 6.0 mM) and

L

-phenylalanine (0–30 mM) as shown, (B) various concentrations of Ca2+ in the presence of 10 mM

L

-phenylalanine or

D

-phenylalanine and (C) various concentrations of Ca2+ (0.5, 2.0 and 6.0 mM) in the absence or presence of various amino acids as shown. The samples were processed by SDS/PAGE and Western blotting as described in the Experimental section. In (A), lanes between the

L

-phenylalanine concentration–response data (lanes 1–4) and low- and high- Ca2+ controls (shown as lanes 5 and 6 from the left) have been deleted from the image. Molecular masses are indicated in kDa.

Figure 5

Figure 5. Comparison of surface expression between the wild-type CaR and CaR mutant S169T

HEK-293 cells were transiently transfected with FLAG-tagged wild-type or S169T mutant CaRs as described in the Experimental section. The cells were then labelled with sulfo-_N_-hydroxysuccinimido-biotin and lysed in the presence of 100 mM iodoacetamide. CaR proteins were immunoprecipitated using anti-FLAG antibody, suspended in SDS/PAGE sample buffer and assayed for protein content. Equivalent amounts of protein were then processed by SDS/PAGE. Western blotting and detection of biotin-labelled proteins was performed using avidin–HRP and ECL® as described in the Experimental section. The arrows indicate the molecular masses of markers in kDa. The lower-molecular-mass form (approx. 160 kDa) is likely to represent monomers of the mature CaR [32]. The higher-molecular-mass species is presumed to correspond to homodimers.

Figure 6

Figure 6. Effects of L-phenylalanine on Ca2+i mobilization in HEK-293 cells expressing the wild-type CaR and on Ca2+i mobilization and ERK1/2 phosphorylation in HEK-293 cells expressing S169T

HEK-293 cells that stably expressed the wild-type CaR (A) or S169T mutant CaR (B) were loaded with fura 2 and investigated for Ca2+o- and

L

-phenylalanine-stimulated Ca2+i mobilization by microfluorimetry. In (A) and (B), the receptor response is expressed in the form of a calibrated cytoplasmic free Ca2+ concentration (in nM). Compared with the wild-type CaR, S169T exhibited markedly impaired sensitivity to Ca2+o that was restored to near-normal Ca2+o-sensitivity by the addition of 10 mM

L

-phenylalanine. (C) Ca2+o concentration–response data for the activation of ERK1/2 as determined by the SureFire assay in the absence and presence of

L

-phenylalanine in HEK-293 cells that stably expressed the S169T mutant. The data in (A) were obtained in nine experiments. The data in (B) and (C) were obtained in six experiments. ○, Control; ▲, 10 mM

L

-phenylalanine.

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