The calcium-sensing receptor (CaSR) is critical for calcium homeostasis and also mediates amino acid (AA) release of hormones including the GI hormones GLP-1 and PYY, with impacts on macronutrient digestion and energy intake. Solved CaSR structures demonstrate binding sites for both AAs and Ca2+.
The CaSR is sensitive to extracellular Ca2+ (Ca2+o) and AAs in various endocrine cells (e.g., parathyroid), in which AAs promote Ca2+i mobilization and modulate hormone secretion. However, CaSR-expressing HEK-293 cells have yielded conflicting results. Thus, fura-2 loaded and perifused HEK-293 cells exhibit AA-stimulated increases in Ca2+I but in static cultures Ca2+o, but not AAs, induce Gq/11-mediated inositol phosphate (IP) turnover.
We asked: ‘What underlies failure of CaSR-mediated AA responses in some models?’ hypothesising that under some conditions, the CaSR’s AA binding site is loaded via an autocrine mechanism rendering the receptor insensitive to AAs but hypersensitive to Ca2+o.
We investigated this hypothesis in HEK-293 cells stably expressing the CaSR, using an adherent format to facilitate washing. In unwashed cells, Ca2+o-induced IP1 and pERK responses were not enhanced by the CaSR-active AA, L-Phe (10 mM), whereas in washed cells, we observed reduced Ca2+o sensitivity enhanced by L-Phe (p < 0.05). Increasing the interval between washing and activation (from 5-60 min), enhanced Ca2+o-stimulated pERK but decreased L-Phe sensitivity, suggesting accumulation of an endogenous ligand. Mass spectrometry led us to identify reduced glutathione (GSH) as a candidate endogenous ligand of the AA site. GSH accumulated in conditioned medium over 30-60 min and inhibition of GSH synthesis with 100µM buthionine sulfoximine suppressed receptor Ca2+o sensitivity and exaggerated L-Phe responsiveness.
Our results support autocrine modulation of the CaSR’s AA binding site by glutathione and provide a novel mechanism by which CaSR-expressing cells tune their responsiveness to Ca2+ and/or AAs.