CD9 is a tetraspan protein that associates with several ␤1 integrins, including ␣6␤1. Because ␣6␤1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin ␤), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin ␤ disintegrin domain. (Both ligands bind to the egg via ␣6␤1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin ␣6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, ␣6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin ␤ ''knockout'' sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin ␤ ؊͞؊ male mice. Our results suggest that high-avidity binding between fertilin ␤ (ADAM 2) and ␣6␤1 requires cooperation between ␣6␤1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as spermegg interactions.

Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin ␤) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D؉C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D؉C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 M, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (ϳ75 versus ϳ55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-␣ 6 monoclonal antibody, GoH3, but not by a nonfunctionblocking anti-␣ 6 monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2. ADAMs1 are a large group of type I integral membrane glycoproteins that contain a disintegrin and a metalloprotease domain (1, 2). Following their metalloprotease and disintegrinlike domains, they contain a cysteine-rich domain, an EGF repeat, a transmembrane domain, and a cytoplasmic tail. Their closest relatives are the P-III snake venom metalloproteases (SVMPs), which are secreted proteins that contain a disintegrin-like and a metalloprotease domain as well as a cysteinerich domain. P-II SVMPs contain metalloprotease and disintegrin domains but lack the cysteine-rich (and other) domains.The disintegrin domains of the P-II SVMPs have a 13-amino acid loop containing, at their tips, sequences such as RGD (kistrin and echistatin), KGD (barbourin), and MVD (atrolysin E). Several P-II snake disintegrins have been shown to bind to the platelet integrin, ␣ IIb ␤ 3, thereby preventing binding of fibrinogen and inhibiting platelet aggregation. Inhibition of platelet aggregation accounts, in part, for the severe hemorrhagic response in snakebite victims. The disintegrin-like domains of P-III SVMPs differ from their P-II counterparts in having two additional cysteine residues and a 14-residue predicted loop that aligns with the 13-amino acid "RGD" loop found in the P-II disintegrins. One of the additional cysteine residues is near the center of the disintegrin loop. The disintegrin-like domain of the P-III SVMP atrolysin A has been characterized. Atrolysin A purified from snake ...

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

We show that Rap2 is involved in androgen-mediated transcriptional and growth responses of human prostate cancer cells.