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Spatiotemporal Interactions of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) Protein with the Actin Cytoskeleton and Exocytosis of Oxytocin upon Prostaglandin F₂ Stimulation of Bovine Luteal Cells¹

Departments of Biochemistry/Biophysics and Animal Sciences,³ Oregon State University, Corvallis, Oregon 97331 Laboratory of Molecular Pharmacology, Biosignal Research Center,⁴ Kobe University, Kobe 657-8501, Japan

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F₂ (PGF₂) 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 PGF₂ treatment and 2) evaluate PGF₂-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, PGF₂ (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 µM). Treatment of cells expressing wild-type MARCKS-GFP with PGF₂ 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 PGF₂ treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.

¹ This research was supported in part by USDA NRICG grant 97-35203-4681 and in part by National Institute of Environmental Health Sciences grant P30 ES00210.

² Correspondence: Fredrick Stormshak, Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331. FAX: 541 737 4174; fred.stormshak{at}orst.edu

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