Effect of cellular expression of pleckstrin homology domains on Gi-coupled receptor signaling.

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.