This renewal application is concerned with the signalling mechanisms by which a sperm causes an egg to begin development at fertilization. The project is focussed on the earliest events of this process, that occur at the egg plasma membrane. These membrane events cause a rise in intracellular Ca2+, which in turn causes many subsequent developmental process. The project includes 3 specific aims: (1) To examine the role of G-alpha-q family proteins in signal transduction at fertilization. The amounts of G-alpha-q family proteins in frog eggs will be reduced by injection of antisense oligonucleotides, to determine if this inhibits Ca2+ release at fertilization. These studies will also examine whether G-alpha-q is activated at fertilization, using protection of the G-alpha-q protein from proteolysis as an assay. (2) To examine the role of tyrosine phosphorylation of phospholipase C-gamma in signal transduction at fertilization. Activation of phospholipase C-gamma will be inhibited by injection of PLC-gamma SH2 domains, to determine if this reduces Ca2+ release at fertilization. These studies will also examine whether PLC-gamma is activated at fertilization, using tyrosine phosphorylation of this enzyme as a assay. (3) To examine the role of alpha-v integrins in signal transduction at fertilization. These studies will investigate whether stimulation of an alpha-v integrin causes a rise in intracellular Ca2+ in amphibian eggs, and whether it increases the amount of the lipid PIP2, the substrate for phospholipase C. They will also examine whether Ca2+ release at fertilization is reduced by inhibition of alpha-v integrin function.
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