In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2α (PGF2α) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2α treatment and 2) evaluate PGF2α-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2α (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 μM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2α and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2α treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.
You have requested a machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Neither BioOne nor the owners and publishers of the content make, and they explicitly disclaim, any express or implied representations or warranties of any kind, including, without limitation, representations and warranties as to the functionality of the translation feature or the accuracy or completeness of the translations.
Translations are not retained in our system. Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the BioOne website.