Progesterone binding at the amphibian oocyte plasma membrane induces meiosis via synthesis of the maturation promoting factor, now recognized to be a universal mitogen. This proposal is concerned with the mechanism of steroid action at the plasma membrane which leads to mitogen synthesis. We have found that the following reactions are triggered by progesterone in isolated oocyte plasma membranes: phosphatidylethanolamine (PE) N-methylation (within seconds), the release of protease and free Ca2+, a rise and fall in 1,2-diacylglycerol (DAG) derived from phosphatidycholine (PC) by phospholipase C (PLC) (within two minutes), activation of protein kinase C (PKC) and increased inositol phosphate turnover. Phospholipid N-methylation, leading to PC synthesis, occurs only within plasma membranes, but 80-90% of the DAG, which activates PKC and is now recognized as a second messenger, is generated from PC. This represents the transduction of a plasma membrane receptor signal to second messenger activation at intracellular membranes. The mechanism by which steroid binding to the plasma membrane receptor activates an enzyme may be G protein-mediated, since our experiments indicate that G proteins directly regulate plasma membrane N- methyltransferase and/or phospholipase C. The first specific aim of this proposal is to use photoaffinity labeling to identify G proteins which are functionally associated with the progesterone receptor; the ultimate goal being to reconstitute the hormone-receptor-G protein system in membrane vesicles.
The second aim i s to determine the molecular species and phospholipid source of the DAGs generated both from plasma membrane and intracellular membranes. About 30% of the total [3H]-glycerol- labeled 1,2--DAG in the prophase oocyte is derived from plasmalogen, a choline phospholipid containing an alpha, beta-unsaturated ether linkage in the 1-position, high in sperm and excitable tissues (brain, heart).
The third aim i s to investigate transient changes in diacylglycerol kinase activity after hormone stimulation; this enzyme controls both DAG levels and the branch point promoting resynthesis of phosphatidylinositols at the expense of resynthesis of the major membrane phospholipids such as PC.
The fourth aim i s to examine the possible role of PKC-dependent protein phosphorylation and/or decreased membrane fluidity in the down-regulation of adenylate cyclase. The proposed research should contribute to the understanding of mitogenic signaling, steroid hormone action at the plasma membrane, and role of phospholipids in generating a second messenger, and the spread of that second messenger signal from plasma to intracellular membranes.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD010463-10A1
Application #
3311303
Study Section
Reproductive Biology Study Section (REB)
Project Start
1977-02-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K (2015) Transmembrane helices in ""classical"" nuclear reproductive steroid receptors: a perspective. Nucl Recept Signal 13:e003
Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K (2015) Computational analysis of the extracellular domain of the Ca²?-sensing receptor: an alternate model for the Ca²? sensing region. Biochem Biophys Res Commun 459:36-41
Morrill, Gene A; Kostellow, Adele B; Moore, Richard D et al. (2013) Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling. BMC Dev Biol 13:3
Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K (2013) A computational analysis of non-genomic plasma membrane progestin binding proteins: signaling through ion channel-linked cell surface receptors. Steroids 78:1233-44
Morrill, Gene A; Kostellow, Adele B; Askari, Amir (2012) Caveolin-Na/K-ATPase interactions: role of transmembrane topology in non-genomic steroid signal transduction. Steroids 77:1160-8
Morrill, Gene A; Dowd, Terry L; Kostellow, Adele B et al. (2011) Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: role of Na/K-ATPase. BMC Dev Biol 11:67
Morrill, Gene A; Kostellow, Adele B; Askari, Amir (2010) Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane. BMC Struct Biol 10:12
Morrill, Gene A; Kostellow, Adele B; Askari, Amir (2008) Progesterone binding to the alpha1-subunit of the Na/K-ATPase on the cell surface: insights from computational modeling. Steroids 73:27-40
Kostellow, Adele B; Morrill, Gene A (2008) Progesterone and subsequent polar metabolites are essential for completion of the first meiotic division in amphibian oocytes. Mol Cell Endocrinol 291:50-6
Morrill, Gene A; Schatz, Frederick; Kostellow, Adele et al. (2006) Gonadotropin stimulation of steroid synthesis and metabolism in the Rana pipiens ovarian follicle: sequential changes in endogenous steroids during ovulation, fertilization and cleavage stages. J Steroid Biochem Mol Biol 99:129-38

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