xestospongin-a and 3-aminobenzeneboronic-acid

Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.

Topics: Active Transport, Cell Nucleus; Animals; Blotting, Western; Boronic Acids; Calcineurin; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cell Nucleus; Enzyme Activation; Enzyme Inhibitors; Estrenes; Gadolinium; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Green Fluorescent Proteins; Humans; Immunohistochemistry; Inositol 1,4,5-Trisphosphate Receptors; Kidney; Lithium Chloride; Luciferases; Macrocyclic Compounds; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; NFATC Transcription Factors; Oxazoles; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Proteins; Pyrrolidinones; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Time Factors; Tissue Distribution; Transfection; TRPP Cation Channels; Type C Phospholipases