Heterotrimetric Gi/o proteins control cyclic AMP oscillations and cytoskeletal structure assembly in primary human granulosa-lutein cells.
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--Gs, Gq, and Gi/o--in primary human granulosa-lutein cells. By exploring the function of Gi/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. Gi/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 Gs, Gi/o, adenylyl cyclases and phosphodiesterase-4. This work supports the paradigm that switching of the agonist-occupied receptors to Gs and Gi/o would control both the intracellular bursts of cAMP (through the gonadotropin-catalysed activation of Gs) and the basal cAMP (through a Gi/o-mediated braking effect).
Laboratoire de Biochimie Hormonale, Groupe Hospitalier Cochin, Paris, France. daniele.saltarelli@cch.ap-hop-paris.fr
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D015152:Blotting, Northern; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D005576:Colforsin; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology*; D019281:Dimerization; D005260:Female; D005455:Fluorescent Antibody Technique; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008184:Luteal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D007986:Luteinizing Hormone / Q000494:pharmacology; D037342:Pertussis Toxin; D013194:Staining and Labeling; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine