The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer.
Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly 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 fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on 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 manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-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 subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding.
Ohlendieck
K
K
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215.
Partin
J S
JS
Lennarz
W J
WJ
D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D005306:Fertilization; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008958:Models, Molecular; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism*; D014817:Vitelline Membrane / Q000378:metabolism
D004220:Disulfides; D006023:Glycoproteins; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor; D004229:Dithiothreitol