Heterotrimeric G i/o Proteins Control Cyclic AMP Oscillations and Cytoskeletal Structure Assembly in Primary Human Granulosa-Lutein Cells

Abstract

In granulosa cells, the luteinising hormone (LH) and the follicle-stimulating hormone (FSH) receptors are coupled to the adenylyl cyclase-cAMP pathway. We identified at least eight different G proteins belonging to three families—G s, G q and G i/o—in primary human granulosa-lutein cells. By exploring the function of G i/o by time-lapse and digital-imaging microscopy of live cells, we found that the reversible actin stress fibre-dependent cytoplasmic retraction of pre-luteinised cells in primary culture is a highly sensitive and quite rapid system allowing detection of an intracellular cAMP surge. This morphology was characterised by maintenance of connexin43-dependent cell–cell contacts and that of microtubule-directed cell processes attached to the substrate and to neighbouring cells. Inhibitors of cyclic nucleotide phosphodiesterase subfamily type 4 (PDE-4), hLH and hFSH provoked this reversible cAMP-dependent phenotype in a temporal-, spatial- and dose-dependent manner. G i/o inhibited adenylyl cyclase in membranes, and cell treatment with islet-activating protein (IAP) caused the cAMP-dependent retracted phenotype. It is concluded that the basal intracellular cAMP level is kept within a narrow range of concentrations, below the threshold for disassembly of stress fibres, through G s, G i/o, adenylyl cyclases and phosphodiesterase-4. This work supports the paradigm that switching of the agonist-occupied receptors to G s and G i/o would control both the intracellular bursts of cAMP (through the gonadotropin-catalysed activation of G s) and the basal cAMP (through a G i/o-mediated braking effect).