Activation of a Gi protein in digitonin/cholate-solubilized membrane preparations of mouse sperm by the zona pellucida, an egg-specific extracellular matrix.
Mammalian sperm possess guanine nucleotide-binding regulatory proteins (G proteins) that are involved in signal transduction pathways leading to zona pellucida (ZP)-mediated acrosomal exocytosis. We have previously examined ZP-G protein dynamics in mouse sperm homogenates, as well as cell-free membrane preparations, and our data support the existence of ZP receptor-G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experimental limitations of the membrane preparations. In the present study, a detergent-solubilized preparation from mouse sperm membranes that retained the signaling properties of cell homogenates and cell-free membrane preparations was developed using buffers containing digitonin and cholate. GTP gamma S, a poorly hydrolyzable analogue of GTP, bound to these solubilized preparations in a specific and concentration-dependent fashion that reached saturation at 100 nM. Incubation of this solubilized membrane preparation with heat-solubilized ZP resulted in an increase in specific GTP gamma S binding in a concentration-dependent manner, with a maximal response at 4-6 ZP/microliters. Mastoparan (50 microM) increased GTP gamma S binding to levels similar to that seen with solubilized ZP. Mastoparan plus ZP stimulated GTP gamma S binding to the same extent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP-stimulated GTP gamma S binding and decreased mastoparan-stimulated GTP gamma S binding by 50-60%. Purified ZP3, the ZP component that possesses quantitatively all of the sperm binding and acrosomal exocytosis-inducing activities of the intact ZP, stimulated GTP gamma S binding to an extent similar to that of solubilized ZP.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia 19104-6080, USA.
Ward
C R
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Kopf
G S
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D000177:Acrosome / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019826:Cholic Acid; D002793:Cholic Acids; D004072:Digitonin; D004527:Egg Proteins / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D010455:Peptides; D011956:Receptors, Cell Surface; D015398:Signal Transduction; D012995:Solubility; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D014862:Wasp Venoms / Q000494:pharmacology; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D002793:Cholic Acids; D004527:Egg Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D011956:Receptors, Cell Surface; D014862:Wasp Venoms; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D019204:GTP-Binding Proteins; D019826:Cholic Acid; D004072:Digitonin