Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation.
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.
Lee
Heather J
HJ
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.
Mun
Hee-Chang
HC
Lewis
Narelle C
NC
Crouch
Michael F
MF
Culverston
Emma L
EL
Mason
Rebecca S
RS
Conigrave
Arthur D
AD
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D007668:Kidney; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism
D000596:Amino Acids; D015220:Calcium Channels; D011994:Recombinant Proteins; D010649:Phenylalanine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3