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.
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