Gαi/o-dependent Ca(2+) mobilization and Gαq-dependent PKCα regulation of Ca(2+)-sensing receptor-mediated responses in N18TG2 neuroblastoma cells.

A functional Ca(2+)-sensing receptor (CaS) is expressed endogenously in mouse N18TG2 neuroblastoma cells, and sequence analysis of the cDNA indicates high homology with both rat and human parathyroid CaS cDNAs. The CaS in N18TG2 cells appears as a single immunoreactive protein band at about 150 kDa on Western blots, consistent with native CaS from dorsal root ganglia. Both wild type (WT) and Gαq antisense knock-down (KD) cells responded to Ca(2+) and calindol, a positive allosteric modulator of the CaS, with a transient increase in intracellular Ca(2+) concentration ([Ca(2+)]i), which was larger in the Gαq KD cells. Stimulation with 1 mM extracellular Ca(2+) (Ca(2+)e) increased [Ca(2+)]i in N18TG2 Gαq KD compared to WT cells. Ca(2+) mobilization was dependent on pertussis toxin-sensitive Gαi/o proteins and reduced by 30 μM 2-amino-ethyldiphenyl borate and 50 μM nifedipine to the same plateau levels in both cell types. Membrane-associated PKCα and p-PKCα increased with increasing [Ca(2+)]e in WT cells, but decreased in Gαq KD cells. Treatment of cells with 1 μM Gӧ 6976, a Ca(2+)-specific PKC inhibitor reduced Ca(2+) mobilization and membrane-associated PKCα and p-PKCα in both cell types. The results indicate that the CaS-mediated increase in [Ca(2+)]i in N18TG2 cells is dependent on Gαi/o proteins via inositol-1,4,5-triphosphate (IP3) channels and store-operated Ca(2+) entry channels, whereas modulation of CaS responses involving PKCα phosphorylation and translocation to the plasma membrane occurs via a Gαq mechanism.