The overall objective of this project is to develop a model system for the study of the mechanism by which egg associated factors modulate sperm cell function and, potentially, regulate fertilization. Speract, a peptide isolated from the jelly coat which surrounds the egg of the sea urchin Strongylocentrotus purpuratus, binds to specific receptors on the plasma membrane of homologous sperm cells and initiates changes in sperm cell behavior and metabolism. Previously, Dr. Dangott identified and purified a 77,000 Mr glycoprotein as the speract receptor. A full length cDNA clone has been isolated which codes for the receptor protein. Using antibodies, nucleic acid probes and recombinant speract receptor proteins now available in his laboratory and others to be prepared, he proposes 1) to express recombinant speract receptors in heterologous cells in order to test and analyze the protein's function as a peptide receptor and signal transducer, 2) recombinant chimeric receptor proteins will be engineered and expressed by standard molecular biological techniques in order to characterize the structural basis for the receptor's peptide binding specificity and interaction with putative components of the signalling system, 3) prepare polyclonal antibodies to the speract receptor in rabbits that will be used to purify the receptor from sperm plasma membranes, and 4) proteins associated with the receptor will be identified, purified and primary amino acid sequence determined. The proteins will be characterized for any biochemical activities and evaluated for any role in possible signal transduction pathways.
