The extracellular calcium-sensing receptor (CaR) plays an important role in whole body calcium homeostasis. Agonists for CaR include extracellular calcium (Ca²⁺_o) and the aminoglycoside antibiotic neomycin and stimulation of CaR results in Ca²⁺_i mobilisation and phosphorylation/activation of extracellular-signal regulated kinase (ERK). Conigrave et al. (2000) have reported that in CaR-transfected HEK-293 cells (CaR-HEK), aromatic L-amino acids act as allosteric activators of the receptor. That is, in the presence of partially activating concentrations of CaR agonist, cotreatment with an aromatic amino acid such as L-Phe or L-Trp increases agonist-induced Ca²⁺_i mobilisation. However, the L-amino acids were only investigated for their effects on Ca²⁺_i mobilisation. Thus in the current study we investigated whether L-Phe or L-Trp can increase CaR agonist-induced ERK activity in HEK cells stably transfected with CaR (a kind gift from NPS Pharmaceuticals, UT, USA). ERK 1,2 phosphorylation was quantified by immunoblotting using a phospho-specific anti-ERK 1,2 polyclonal antibody. All experiments were performed at 37 °C for 5 min in an experimental buffer containing 0.5 mM Ca²⁺ (unless otherwise stated). Data are from a minimum of three independent experiments.

A partially activating concentration of neomycin (30 mM) stimulated ERK 1,2 phophorylation 3.7-fold (± 1.5, S.E.M.) above control, an effect which was submaximal since 100 mM neomycin induced a 38.8-fold (± 14.1) stimulation of ERK. However, when cotreated with 30 mM neomycin, L-Phe (10 mM) did not increase the stimulatory effect of neomycin on its own (Neo and L-Phe, +1.7 ± 0.4-fold). This lack of effect of L-Phe was not agonist specific, since when 2.5 mM Ca²⁺_o was employed, L-Phe failed to increase the Ca²⁺_o-induced response (Ca²⁺_o, +10.5 ± 5.2-fold; Ca²⁺_o and L-Phe, +8.4 ± 3.2-fold). Again, the response to 2.5 mM Ca²⁺_o was not maximal since 5 mM Ca²⁺_o caused a 130-fold (± 45.1) stimulation of ERK. Next, we tested whether the lack of effect was amino acid specific. However, L-Trp (10 mM) also failed to increase 30 mM neomycin-induced ERK activation (Neo, +7.6 ± 4.7-fold; Neo and L-Trp, +5.8 ± 1.8-fold) or 2.5 mM Ca²⁺_o-induced ERK activation (Ca²⁺_o, 4.4 ± 2.2-fold; Ca²⁺_o and L-Trp 7.6 ± 3.4-fold ). Again, increasing the CaR agonist concentration in the absence of L-Trp caused substantial elevations in ERK activity (100 mM neomycin, +249.6 ± 171-fold; 5 mM Ca²⁺_o, +68 ± 25.2-fold). In all experiments, the addition of L-Phe or L-Trp (10 mM) alone had no effect on basal ERK phosphorylation.

Thus the aromatic amino acids L-Phe and L-Trp do not stimulate the CaR to induce ERK activation. If aromatic amino acids are allosteric activators of CaR, so eliciting Ca²⁺_i mobilisation, then the cell must somehow differentiate between amino acid and ‘classic’ agonist-induced stimuli to effect differential intracellular signalling. It will be necessary to investigate the reported effects of L-amino acids on CaR using cells that endogenously express the receptor and by measuring a variety of intracellular signals and physiological responses.

The Wellcome Trust supported this work. D.W. is an NKRF Training Fellow.