Modulation of human mineralocorticoid receptor function by protein kinase A

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcriptio n factor modulating specific gene expression in sodium-transporting epithel ia. Physiological evidence suggest a crosstalk between the cAMP- and aldost erone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in Hep G2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulat es glucocorticoid response element (GRE)-containing promoters in a ligand-i ndependent manner. This effect was strictly MR dependent since no activatio n of the reporter gene was observed in the absence of cotransfected hMR exp ression plasmid. Furthermore, a synergistic activation was achieved when ce lls were treated with both aldosterone and cAMP, This synergistic effect wa s also observed in the CV1 and the stable hMR-expressing M cells but was de pendent on the promoter used, In particular, synergism was less pronounced in promoters containing several GREs, We show that (protein kinase-inhibiti ng peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and ald osterone induction, which indicates that a functional cAMP pathway is requi red for stimulation of transcription by aldosterone. Using MR-enriched bacu lovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA, Increased DNA binding of hMR is likely to reflect an increase in the number of active rec eptors, as measured by Scatchard analysis, Using a truncated MR, we show th at the N-terminal domain is required for the effect. Finally, the N-termina l truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repres sor.