PARENT SESSION
MECHANISMS OF HORMONE ACTION
Harborside C
7:30 AM-10:00 AM

(585) SPATIO-TEMPORAL INTERACTIONS OF MARCKS PROTEIN WITH THE ACTIN CYTOSKELETON AND EXOCYTOSIS OF OXYTOCIN UPON PROSTAGLANDIN F₂ STIMULATION OF BOVINE LUTEAL CELLS.

Salli, Ugur¹, Saito, Naoaki², Stormshak, Fredrick¹, ¹ Departments of Biochemistry/Biophysics and Animal Sciences, Corvallis, OR² Laboratory of Molecular Pharmacology, Kobe, Japan

ABSTRACT- In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to PGF₂ is correlated with the secretion of oxytocin (OT). The present study was conducted to 1) determine whether large luteal cells contain MARCKS protein, 2) examine the intracellular translocation of the green fluorescent protein (GFP) conjugated MARCKS (MARCKS-GFP) after PGF₂ treatment and 3) evaluate PGF₂-induced temporal changes in MARCKS-GFP and filamentous actin cortex associated with exocytosis of oxytocin. In Exp. 1, bovine luteal cells were treated with either ethanol (10 l) or PGF₂ (56 nM) for 10 min and then fixed for immunocytochemistry. Localization of phosphorylated MARCKS that was identified by the use of anti-phospho-MARCKS antibody was observed in large luteal cells. In Exp. 2, one wild type (wt) and two mutant MARCKS-GFP constructs were transfected into luteal cells and expression detected through fluorescence microscopy. Forty-eight hours after transfection, luteal cells were treated with ethanol, PGF₂ or phorbol ester, TPA (1 M). Treatment of cells expressing wt MARCKS-GFP with PGF₂ and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. The phosphorylation mutant MARCKS-GFP was found localized mainly on the plasma membrane and treatments did not cause its translocation from the plasma membrane to the cytoplasm. The myristoylation mutant MARCKS-GFP was observed solely in the cytoplasm. There were no changes detected in intracellular location of the myristoylation mutant MARCKS-GFP after treatment. In Exp. 3, luteal cells were transfected with either one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Only large luteal cells that contained positive signals for MARCKS-GFPs, oxytocin and actin filaments were recorded for later analysis. Exp. 3 revealed that only wt MARCKS-GFP transfected 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. Research supported by USDA NRICGP Grant 97-35203-4681.

KEY WORDS: Corpus Luteum, Oxytocin, MARCKS