THE BIOLOGICALLY-ACTIVE FORM OF THE SEA-URCHIN EGG RECEPTOR FOR SPERMIS A DISULFIDE-BONDED HOMO-MULTIMER

Since many cell surface receptors exist in their active form as oligom eric complexes, we have investigated the subunit composition of the bi ologically active sperm receptor in egg plasma membranes from Strongyl ocentrotus purpuratus. Electrophoretic analysis of the receptor withou t prior reduction of disulfide bonds revealed that the surface recepto r exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact tha t the NH2-terminus of the extracellular domain of the sperm receptor i s rich in cysteine residues. Studies with cross-linking agents of vari ous length and hydrophobicity suggest that no other major protein is t ightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertiliz ation was found to be inhibited by treatment of eggs with 5 mM dithiot hreitol, we undertook more direct studies of the effect of reduction o n properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose-dependent m anner, the reduced and alkylated receptor was inactive. Second, we tes ted the ability of the isolated receptor to mediate binding of acrosom e-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunit s and that these must be linked as a multimer via disulfide bonds to p roduce a complex that is functional in sperm recognition and binding.