In the present application, the investigator intends to focus on human zona receptor kinase (ZRK) with particular emphasis on the function of the cytoplasmic domain and its relation to induction of acrosomal exocytosis. The interaction of ZP3 with recombinant ZRK expressed in cultured cells will be explored. In addition to binding studies, the applicant will test whether ZP3 activates expressed ZRK using kinase assays. Signalling pathways that are activated by ZRK in sperm will be analyzed. Studies will be carried out to investigate the role of phospholipase Cg and phosphatidyl inositol 3-kinase in ZP3-regulated acrosome reaction triggering and determine whether other candidate SH2-containing proteins function in ZP-3 stimulated acrosomal exocytosis. The relationship between PTK- and G-protein-regulated pathways, both of which appear to be involved in ZP-triggered acrosomal exocytosis will be dissected. The P.I. will examine protein tyrosine phosphatases in sperm to determine their relationship to ZRK activation and signalling. The yeast two-hybrid system will be employed as an alternative strategy to identify signalling proteins that bind to ZRK, since this method will not introduce the bias of focussing exclusively on phosphotyrosine-mediated protein-protein interactions. Sites in ZRK's cytoplasmic domain involved in binding to signalling components will be mapped by generating GST fusion proteins of the ZRK intracellular domain containing wild-type or mutated sequences. These constructs will be analyzed for their ability to bind downstream signalling proteins of interest and thereby define which residues in the cytoplasmic domain are involved in these interactions. To investigate the cytoplasmic sequences of ZRK that stimulate exocytosis, rat basophilic leukemia cells will be transfected with ZRK and regulated secretion following ZP3 stimulation will be determined. ZRK constructs containing intracellular domain mutations will be used to determine the role of specific ZRK residues/sequences in regulated exocytosis. It is anticipated that these studies will elucidate fundamental properties of the signalling mechanisms that lead to acrosomal exocytosis.
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