A 130-kDa membrane protein of sperm flagella is the receptor for asterosaps, sperm-activating peptides of starfish Asterias amurensis

Spermatozoa of the starfish, Asterias amurensis, have a specific receptor f or asterosap, a sperm-activating peptide isolated from the jelly coat of ho mologous eggs. We characterized the receptor by using several asterosap der ivatives. Analysis of equilibrium binding of radioactive di-iodinated Bolto n-Hunter reagent-labeled asterosap (I-125(2)-BHP15) to the spermatozoa indi cated that the cell has 1.1 x 10(5) binding sites of high affinity (K-d = 5 7 pM), and also the receptor showed positive cooperativity for asterosap bi nding. When spermatozoa were treated with fluorophore-labeled asterosap, th e sperm flagella were labeled, indicating that the receptors are mostly loc alized in the sperm tail. When spermatozoa were reacted with radioactive as terosap prelabeled with photoaffinity cross-linkers, a single 130-kDa membr ane protein of sperm flagella was specifically radiolabeled. This result wa s reproducible regardless of the length of spacer arm of cross-linkers so f ar studied. Therefore, the 130-kDa protein is likely to be the receptor for asterosaps. Modification of asterosap at the N-terminal region with bulky molecules such as carboxyfluorescein did not affect the activity of asteros ap, suggesting that the N-terminus of asterosap is not involved in the liga nd-receptor interaction. On the other hand, S-alkylated asterosaps did not compete with I-125(2)-BHP15 for binding to the receptor, indicating that di sulfide linkage of asterosap is essential for the ligand-receptor interacti on. The properties of the receptor, high affinity and high concentration, e nabled us to apply the fluorescence polarization technique to study the mol ecular interaction between asterosap and the receptor. Using this method, w e performed binding experiments in almost real time and found that divalent cations are significantly involved in the interaction between asterosap an d the receptor. (C) 2000 Academic Press.