Beta gamma subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C.

Abstract

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an .alpha. subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the .beta. subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the .beta. subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte .beta..gamma.-subunit complex or 1.0 ng of bovine brain .beta..gamma.-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled .beta..gamma. subunits, bovine serum albumin, or resolved .alpha. subunits also showed no reduction in response. Cells injected with various concentrations of .beta..gamma. subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain .beta..gamma. subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-triphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an .alpha..beta..gamma. heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.