Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release.
Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
M
M
Bai
S
S
Trivedi
C R
CR
Lane
Y
Y
Yang
S J
SJ
Quinn
E M
EM
Brown
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D010281:Parathyroid Hormone; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D013912:Threonine; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium