* Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908; and Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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Integrins can exist in different functional
states with low or high binding capacity for particular
ligands. We previously provided evidence that the integrin 6
1, on mouse eggs and on
6-transfected cells,
interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin
). In the present study
we tested the hypothesis that different states of
6
1
interact with fertilin and laminin, an extracellular matrix ligand for
6
1. Using
6-transfected cells we
found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding.
Next, we compared the ability of fluorescent beads
coated with either fertilin
or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin
beads bound to eggs via an interaction mediated by
the disintegrin loop of fertilin
and by the
6 integrin subunit. In Ca2+-containing media, laminin E8 beads
did not bind to eggs. Treatment of eggs with phorbol
myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not
induce laminin E8 bead binding. Treatment of eggs
with Mn2+ dramatically increased laminin E8 bead
binding, and inhibited fertilin bead binding. Our results
provide the first evidence that different states of an integrin (
6
1) can interact with an extracellular matrix
ligand (laminin) or a membrane-anchored cell surface
ligand (ADAM 2).
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Introduction |
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INTEGRINS are transmembrane heterodimers that act as
receptors for extracellular matrix components and
membrane-anchored cell surface coreceptors (Hynes
and Lander, 1992; Clark and Brugge, 1995
). Integrins can
be activated to bind extracellular ligands by intracellular
signals. Such inside-out signaling is thought to involve
changes in the conformation and/or aggregation state of
the integrin (Faull and Ginsberg, 1995
; Brown and Hogg,
1996
; Bazzoni and Hemler, 1998
; Hato et al., 1998
). Inside-out signaling can be invoked experimentally by activating
the protein kinase C pathway with phorbol esters (Strulovici et al., 1991
) or by coexpressing certain integrin-associated proteins or oncogenes (Fenczik et al., 1997
; Hughes
et al., 1997
; Hemler, 1998
; Porter and Hogg, 1998
). Integrins can also be activated by several extracellular mechanisms including: varying divalent cations, applying certain ligands or antibodies, and controlling whether or not the
subunit is proteolytically processed (Delwel et al., 1996
).
Whether activated by intracellular or extracellular means,
what has been observed to date is activation of an integrin from a low to a high binding state for a particular
ligand(s), or differential binding of specific integrin antibodies to the low and high binding states (Diamond and
Springer, 1994
; van Kooyk et al., 1994
; Faull and Ginsberg, 1995
).
Fertilin and
are sperm surface glycoproteins that
have been implicated in binding and fusion to the egg
plasma membrane (Blobel et al., 1992
; Bigler et al., 1997
;
Cho et al., 1998
). Fertilin
and
(ADAM 1 and 2) are
also the prototypes of the ADAM1 family of cell surface
proteins (Wolfsberg and White, 1996
; Black and White,
1998
). ADAMs have been referred to also as MDCs, cellular disintegrins, metalloprotease-disintegrins, and disintegrin-metalloproteases. A table of the ADAMs is available
at http://www.med.virginia.edu/~jag6n/whitelab.html. Like
their closest relatives, the snake venom metalloproteases (SVMPs), ADAMs contain disintegrin and metalloprotease domains. Several snake venom disintegrins have
been shown to interact with integrins including
IIb
3 and
2
1 (DeLuca et al., 1995
; Jia et al., 1996
; Kamiguti et al.,
1996
). Fertilin
and
are proteolytically processed during
sperm maturation such that on mature fertilization competent sperm (the prodomain and metalloprotease domain
are removed) each subunit begins with its disintegrin domain (Lum and Blobel, 1997
; Waters and White, 1997
;
Bigler et al., 1997
). The disintegrin domain of fertilin
has
been implicated as a key, albeit not exclusive, participant
in sperm-egg binding based on the inhibitory action of
peptide analogues of the disintegrin loop (Myles et al., 1994
;
Almeida et al., 1995
; Evans et al., 1995
; Gichuhi et al., 1997
),
antibodies against the disintegrin loop (Yuan et al., 1997
), and
recombinant proteins containing the disintegrin domain
(Evans et al., 1997
; Bigler, D., M.S. Chen, Y. Takahashi,
E.A.C. Almeida, and J.M. White, manuscript in preparation). Further studies have demonstrated that the integrin
6, presumably as the
6
1 complex (Almeida et al., 1995
),
is involved in fertilin-mediated sperm binding. Antibodies
to the
6 subunit bind to zona-free mouse eggs and inhibit
sperm binding. In addition, cells that express the
6 and
1 integrin subunits bind more sperm than their nonexpressing counterparts (Almeida et al., 1995
; Chen, M.S., and J.M. White, unpublished data).
6
1 is a well characterized laminin receptor (Mercurio and Shaw, 1991
; Ekblom, 1996
). Therefore, it may participate in both cell-cell
interactions, via the disintegrin domain of an ADAM, as
well as cell-extracellular matrix (ECM) interactions, by
engaging laminin.
Like many integrins, 6
1 on cultured cells appears to
rest in a basal state for adhering to laminin. The ability of
6
1 to bind laminin can be stimulated two- to fivefold by
treating cells with phorbol esters or by adding Mn2+ to the
extracellular medium. Activation occurs without a change in the amount of
6 at the cell surface, suggesting that
modulation is caused by changes in either the conformation (affinity) and/or aggregation (avidity) state of the integrin complex (Shaw et al., 1993
; Shaw and Mercurio,
1993
). These observations suggest, as with
IIb
3 and
other integrins, that adhesion of
6
1 to laminin is responsive to physiological stimulation. The major goal of this
study was to determine how agents that modulate the ability of
6
1 to interact with laminin affect binding of sperm
and, more specifically, the ADAM protein, fertilin
.
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Materials and Methods |
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Egg Isolation
Mature oocytes were collected from 8-12-wk-old ICR female mice (Harlan Sprague Dawley Inc.) as described previously (Almeida et al., 1995).
To prepare zona-free eggs, egg masses were incubated with 3 mg/ml hyaluronidase () in embryo culture medium (TE) (Spindle, 1980
) for 5-10 min at 37°C to dissociate cumulus cells, and washed
through three 200-µl drops of fresh TE. Eggs were further treated with 10 µg/ml of
-chymotrypsin () in TE for 3 min at 37°C to
soften the zona pellucida. When the perivitelline space was visibly enlarged, eggs were immediately washed through two 200-µl drops of fresh TE. The loosened zonae were mechanically removed by gently passing eggs through a glass pipette ~100 µm in diameter. Eggs were placed in
TE, overlaid with light mineral oil, and incubated for 1 h at 37°C in a 5%
CO2 incubator before use. Alternatively, where indicated, egg masses
were collected in M199 culture medium () supplemented with
0.4% BSA (fraction V, fatty acid free; ), 3.5 mM pyruvate (), 100 U/ml penicillin G sodium (), and 100 µg/ml streptomycin sulfate (). The egg masses were then incubated with 0.6 mg/ml hyaluronidase for 3 min, washed through three 100-µl drops of fresh M199, treated for 1 min in acidic Tyrode's solution, pH
2.5 (Hogan et al., 1994
), washed through three 100-µl drops of fresh M199,
and incubated in M199 for 3 h at 37°C in a 5% CO2 incubator before use.
We have found that a 3-h resting period after a brief incubation in acidic Tyrode's solution is necessary for the recovery of egg surface proteins, including the
6 integrin subunit, and for recovery of optimal fertilization
competence (Takahashi et al., 1995
). Experiments using eggs treated either with chymotrypsin followed by mechanical shearing and a 1-h recovery, or with acidic Tyrode's solution (followed by a 3-h recovery) to remove the zona pellucida gave similar results.
Sperm Isolation and Solubilization
Sperm were isolated from the cauda epididymis and vas deferens of ICR
retired male breeders (Harlan Sprague Dawley Inc.; Hilltop) as described
previously (Almeida et al., 1995). For the majority of experiments sperm
were capacitated for 2 h by incubation in TE/3% BSA in a CO2 incubator
at 37°C. At this time ~50% of the sperm had undergone the acrosome reaction. To prepare sperm lysates for coating fluorescent beads, sperm
were washed twice with Dulbecco's phosphate-buffered saline (PBS;
) and resuspended at 5 × 106 sperm in 0.25 ml lysis buffer
(CHAPS/gelsolin) at 4°C. CHAPS/gelsolin contains 1.5% CHAPS (), 10 mM Tris (pH 7.4), 50 mM NaCl, 1 mM MgCl, 1 mM
CaCl2, 5 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin A, 10 µg/ml
leupeptin, 20 µg/ml aprotonin, 50 µg/ml antipain, 2 mM benzamidine, 50 µg/ml soybean trypsin inhibitor, 2.5 mM iodoacetamide, and 50 µg/ml
gelsolin (gift of Dr. P. Janmey; Cytoskeleton). The sperm suspension was
passed four times through a syringe with a 27-gauge needle and incubated
for 4-18 h at 4°C. Lysates were centrifuged at 14,000 rpm for 10 min at
4°C, and the supernatant was passed through an 0.2-µm syringe filter. For most experiments the pellet was reextracted with another 0.25 ml of
CHAPS/gelsolin, passed four times through a syringe, and incubated for
4 h at 4°C. This lysate was collected, clarified by centrifugation (as above),
and pooled with the original extract.
Antibodies
Antibodies were obtained from the following sources: rat anti-6 mAb
GoH3 (Immunotech) and rat anti-
6 mAb J1b5 (Dr. C. Damsky). A polyclonal rabbit antiserum against fertilin
was raised against a peptide analogue of its cytoplasmic domain. The polyclonal rabbit antiserum (anti-envelope [ENV]) against the cytoplasmic domain of the Avian Leukosis
and Sarcoma Virus subtype C envelope glycoprotein was described previously (Gilbert et al., 1994
). Polyclonal antibodies were purified on a column of the immunizing peptide coupled to a gel (SulfoLink Coupling Gel;
). Antibodies were eluted with 100 mM glycine, pH
2.5, immediately neutralized with 1 M Tris-HCl, and dialyzed extensively
with PBS.
Peptides
14-mer peptides corresponding to the sequence of the predicted binding
loop of the disintegrin domain of mouse fertilin , as well as a scrambled
fertilin
sequence, were synthesized on a peptide synthesizer (Symphony;
Protein Technologies) and purified by HPLC. The following sequences
were used: fertilin
, CRLAQDEADVTEYC; and scrambled fertilin
,
CETADYQRVECLDC. Peptides were amidated at the COOH terminus
and acetylated at the NH2 terminus. The two terminal cysteine residues
were protected with acetoamidomethyl groups. Peptides were dissolved in
DMSO to a concentration of 25 mM, and diluted to 250 µM in egg medium immediately before use.
Preparation of Protein-coated Fluorescent Beads
0.2 µm yellow-green or crimson fluorescent sulfate-derivatized latex
beads (Molecular Probes, Inc.) were coated with fertilin or laminin E8
as follows. To prepare fertilin
-coated beads, beads from 10 µl of a 2%
bead suspension were incubated with 10 µl of anti-fertilin
cytoplasmic
tail antibody (0.42 mg/ml) for 4 h at 4°C on an orbital platform mixer
(Clay Adams). Beads were washed twice with PBS, and incubated overnight with sperm lysates prepared as described above. To prepare beads
coated with laminin E8 (gift of Dr. P. Yurchenco), beads were incubated
with a solution of laminin E8 (1 mg/ml in PBS) for 2 to 3 h at 4°C on an orbital platform mixer. Fertilin- or laminin E8-coated beads were washed twice with PBS, quenched for 1 h with 0.2 mg/ml goat anti-rabbit IgG
(), washed twice with PBS, and resuspended to 0.2%
in PBS. Beads were used on the day of preparation and were sonicated in
a water bath sonicator (Laboratory Supplies) three times for 5 s each at
4°C immediately before use.
Bead-Egg Binding Assay
20-40 zona-free eggs, prepared as described above, were pretreated for 15 min with experimental agents in 20-µl drops of TE. The divalent cation composition of TE is 2.4 mM Ca2+, 0.47 mM Mg2+. Protein-coated fluorescent beads were added to give a final concentration of 0.02%, and eggs were incubated in a 5% CO2 incubator. Alternately, where indicated, eggs were pretreated for 15 min with experimental agents, and incubated with beads in Puck's saline A (), a buffer that does not contain any divalent cations. Where indicated, eggs were pretreated with latrunculin A (Biomol) for 1 h in M199, washed three times with Puck's saline A, and incubated with beads as described above. The eggs were gently agitated every 15 min. After 1 h at 37°C, the eggs were washed with three 100-µl drops of fresh medium using an ~100 µm glass pipette. After washes, small drops containing the eggs were placed in 24-well dishes, and overlaid with light mineral oil for imaging by confocal microscopy.
Sperm-Macrophage Binding Assay
6B- and mock-transfected P388D1 cells were previously generated by
transfection of cells with the pRc/CMV vector containing the human
6B
cDNA or vector alone; the vector alone contains the neomycin resistance
gene (Shaw et al., 1993
). Cells transfected with the vector alone are referred to in this paper as "mock-transfected." Subconfluent monolayers of
6B- or mock-transfected P388D1 macrophages were grown in RPMI
1640 () supplemented with 15% heat-inactivated fetal bovine
serum (HyClone Laboratories Inc.) 2 mM glutamine (), 25 mM
Hepes, pH 7.4, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 0.3 mg/ml Geneticin (G418 sulfate; ). Cells were detached from culture dishes by scraping and resuspended in RPMI to a concentration of 105 cells/ml. 5 × 104 cells were plated in 24- or 48-well tissue
culture dishes (Falcon Plastics) for 3 h at 37°C in a 5% CO2 incubator. The
cells were washed twice with TE/3% BSA and pretreated with experimental agents for 15 min at 37°C. Capacitated sperm were added to a final
concentration of 5 × 106 sperm/ml, and incubated for 1 h at 37°C with gentle shaking at 15-min intervals in a 5% CO2 incubator in the presence of
experimental agents. Cells were washed once with TE/3% BSA, once with
PBS, and fixed with 1.6% paraformaldehyde in PBS. The cultures were observed with a phase-contrast microscope and the number of sperm bound per cell was counted. Five fields per well of duplicate wells were
analyzed for each condition. Each field contained ~100 cells. Cation dependence experiments were performed in Puck's saline A instead of
RPMI. Cells from the same starting suspension were used for parallel
sperm-macrophage binding assays and macrophage-laminin adhesion assays.
Macrophage-Laminin Adhesion Assay
Laminin adhesion assays were performed as described previously (Shaw
et al., 1993) and in parallel with sperm-macrophage binding assays.
Briefly, 96-well plates (Costar Plastics) were coated for 14-18 h with 20 µg/ml EHS laminin-1 () in Ca2+-, Mg2+-free PBS
(), and washed three times with PBS before use. Subconfluent
monolayers of transfected P388D1 macrophages were washed once with
RPMI, detached from culture dishes by scraping, and resuspended in
RPMI to a concentration of 5-10 × 105 cells/ml. Cell suspensions were
added to the laminin-1-coated wells, and incubated for 1 h in the presence
of experimental agents as indicated. Cells were washed twice with RPMI,
fixed with methanol for 15 min at 25°C, stained with 0.2% crystal violet in
2% ethanol for 15 min, washed several times with water, and solubilized
with 1% SDS for 1 h. Optical density was measured at 595 nm using a microplate reader (Molecular Devices Corp.). Quadruplicate samples were
analyzed for each data point. Cation dependence experiments were performed in Puck's saline A instead of RPMI.
Sperm-Egg Binding Assay
Zona-free eggs were placed in 100-µl drops of TE under mineral oil or, where indicated, in drops of Puck's saline A supplemented with the indicated amount of CaCl2. Eggs were pretreated with experimental agents for 15-30 min before sperm addition. Capacitated sperm were added to give a final concentration of ~5 × 105 sperm/ml, and incubated for 1 h at 37°C in a 5% CO2 incubator. Eggs were washed three times in fresh medium, fixed in 4% glutaraldehyde, and mounted on glass slides for observation by phase-contrast microscopy. 30-40 eggs were analyzed per condition and the average number of sperm bound per egg was determined.
Western Blotting, Biotinylation, and Immunoprecipitation of Sperm Proteins
For Western blots, sperm from the caput or cauda epididymis were isolated in M199 containing 0.1% polyvinyl alcohol ()
and, unless indicated, lysed in CHAPS/gelsolin as described above. The
lysate was incubated with 50 µl of a 50% slurry of concanavalin A (ConA)
agarose beads (Vector Laboratories Inc.) for 1 h at 4°C. The beads were
washed twice with PBS, resuspended in 15 µl of 2× SDS gel sample buffer
(250 mM Tris-HCl, pH 7, 12.5% sucrose, 8% SDS, 20 mM EDTA, 0.2 mg/ml bromophenol blue, and where indicated, 100 mM dithiothreitol),
incubated at 95°C for 5 min, and then subjected to 10% polyacrylamide
gel electrophoresis. Proteins were transferred to nitrocellulose (Schleicher
and Schuell Inc.) and the membrane was incubated with blocking buffer
(1 M glucose, 10% glycerol, 3% BSA, 1% milk, and 0.5% Tween 20 in
PBS, pH 7.4) for 30 min. The nitrocellulose was then incubated with 5 µg/ml
purified anti-fertilin cytoplasmic tail antibody in TBST (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20 []) for 14 h
at 4°C followed by incubation with goat anti-rabbit IgG coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc.) in TBST
for 1 h. Horseradish peroxidase was detected by enhanced chemiluminescence.
For immunoprecipitation of fertilin from solubilized sperm, ~1 million capacitated sperm from the cauda epididymis were incubated with 1 mg/ml EZ-Link sulfo-NHS-LC-biotin () for 15 min at
25°C, washed twice with PBS, and lysed in CHAPS/gelsolin as described
above. Supernatants were precleared for 1 h at 4°C with 10 µl of protein A
agarose beads () coupled with preimmune
IgG (3 µl serum/10 µl beads) for both the anti-fertilin
, and anti-ENV
antibodies. Supernatants were divided and immunoprecipitated for 1 h at
4°C with 5 µl of protein A agarose beads coupled with either anti-fertilin
or anti-ENV IgGs (0.2 µg/µl beads). Beads were divided and washed either twice with PBS, or seven times with RIPA buffer (10 mM Tris-HCl,
pH 7.4, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM
NaCl). Beads were resuspended in 15 µl of 2× SDS gel sample buffer, incubated at 95°C for 5 min, and subjected to nonreducing 10% polyacrylamide gel electrophoresis. Samples were transferred to nitrocellulose, and
biotinylated proteins were detected (Vectastain ABC Elite; Vector Laboratories Inc.) by enhanced chemiluminescence.
Staining of Zona-free Eggs with FITC-Phalloidin
Zona-free eggs were prepared using acidic Tyrode's solution as described above and allowed to recover for 3 h at 37°C. Where indicated, eggs were treated for 1 h with 20 µg/ml latrunculin A in M199. Eggs were fixed with 1% paraformaldehyde (Electron Microscopy Sciences), and 0.1% polyvinyl alcohol (), in PBS for 30 min at 25°C. Eggs were washed three times with blocking buffer (20 mM glycine and 2% BSA in PBS) and permeabilized with 2% Triton X-100 () in blocking buffer for 1 h at 25°C. Eggs were washed three times with blocking buffer, and incubated with 100 nM FITC-phalloidin () in blocking buffer for 30 min at 25°C. Eggs were washed four times with blocking buffer and mounted onto slides for analysis by confocal microscopy.
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Results |
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We previously provided evidence that the disintegrin domain of fertilin (ADAM 2) can interact with the integrin
6
1. An anti-
6 mAb (GoH3) as well as peptide analogues of the fertilin
disintegrin loop specifically inhibited sperm-egg binding. Sperm bound more extensively to
cells that expressed
6
1 than to their nonexpressing
counterparts. This binding was specifically inhibited by
GoH3 and by a fertilin
disintegrin loop peptide analogue (Almeida et al., 1995
). Since
6
1 is a well characterized
laminin receptor whose apparent avidity for laminin can
be regulated experimentally, we compared the effects of
agents that modify the apparent avidity of
6
1 for laminin for their effects on binding of the ADAM protein, fertilin
. We first compared the effects of integrin avidity/ affinity modulators on sperm and laminin binding to
6-transfected cells. We also compared their effects on the
ability of eggs to bind beads coated with either fertilin or
laminin E8.
Inverse Effects of Integrin Avidity/Affinity Modulators on Binding of Sperm and Laminin
When transfected with the 6 subunit cDNA P388D1 mouse
macrophages express
6 in complex with a protein the size
of the
1 integrin subunit on the cell surface. No protein
the size of the
4 subunit, the only other known
6 binding
partner (Sonnenberg et al., 1987
; Hemler et al., 1989
), was
seen (Shaw et al., 1993
). As shown previously, (Shaw et al.,
1993
) and in Fig. 1 a, left,
6-transfected macrophages adhered to laminin to a greater extent than their mock-transfected counterparts. This adhesion could be stimulated by
treatment with PMA. As shown previously (Almeida et al.,
1995
) and in Fig. 1 a, right,
6-transfected macrophages bound more sperm than their mock-transfected counterparts. However, in contrast to its enhancing effect on laminin adhesion, PMA inhibited sperm binding to parallel
cultures of
6-transfected macrophages (Fig. 1 a, right).
As observed previously (Shaw, L., and A. Mercurio, unpublished data), genistein, a tyrosine kinase inhibitor,
reversed the PMA stimulation of laminin adhesion to
6-transfected macrophages (Fig. 1 b, left). In contrast, genistein restored sperm binding to parallel cultures of PMA-treated
6-transfected macrophages (Fig. 1 b, right).
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Inclusion of Mn2+ in the extracellular medium has also
been shown to modulate integrin function. As shown
previously (Shaw et al., 1993) and in Fig. 2 a, left, Mn2+
stimulated the adhesion of
6-transfected macrophages to
laminin. In contrast, Mn2+ inhibited sperm binding to
6-transfected macrophages (Fig. 2 a, right). The number of
sperm bound at 0.01 mM Mn2+ was less than at 0.05 mM
Mn2+, in accordance with previous observations that some
amount of a divalent cation is required for sperm binding
to eggs (Yanagimachi, 1978
; Evans et al., 1995
). When
6-transfected macrophages were incubated in medium containing 0.5 mM Mn2+ and increasing amounts of Ca2+,
their ability to adhere to laminin decreased (Shaw and
Mercurio, 1994
) (Fig. 2 b, left). Conversely, adding increasing amounts of Ca2+ to medium containing 0.5 mM
Mn2+ increased sperm binding to
6-transfected macrophages (Fig. 2 b, right). Adhesion of mock-transfected
cells to laminin was not increased in the presence of Mn2+
(Shaw et al., 1993
; Chen, M., and J. White, unpublished
data).
|
Collectively, the results presented in Figs. 1 and 2 indicate that PMA and Mn2+ stimulate laminin adhesion to
6-transfected macrophages, whereas PMA and Mn2+ inhibit sperm binding to these cells. Reciprocally, whereas
genistein and Ca2+ inhibit the stimulatory effects of PMA
and Mn2+, respectively, on laminin adhesion, they restore
sperm binding to PMA- and Mn2+-treated cultures of
6-transfected macrophages. Neither PMA nor Mn2+ visibly
affected sperm motility (Chen, M., E. Almeida, and J. White, unpublished data).
We next tested the effects of PMA and Mn2+ on sperm
binding to zona-free mouse eggs. As seen in Fig. 3 a, PMA
inhibited sperm binding. Under the conditions of this experiment, we did not see evidence of egg activation, for example cortical granule exocytosis (Almeida, E., and J. White, unpublished data). Addition of Mn2+ to our basic
egg medium (that contains 2.4 mM Ca2+) inhibited sperm
binding (Fig. 3 b). When eggs were placed in a medium
free of divalent cations and supplemented with CaCl2, we
found that Ca2+ supported sperm binding. This finding
corroborates previous observations that Ca2+ (~1.8 mM)
is needed for optimal sperm binding (Fig. 3 c; for reviews
see Yanagimachi, 1978; Fraser, 1994
). We were unable to test the effect of genistein on sperm-egg binding because
at the concentrations used for the
6-expressing cells (Fig.
1 b, right), the eggs lysed.
|
Binding of Fertilin-coated Beads to Eggs
We next further probed the molecular basis for the apparent inverse effects of integrin avidity/affinity modulators
on 6-mediated binding of sperm and laminin. Since our
hypothesis is that fertilin
(ADAM 2) is responsible, at
least in part, for sperm binding to
6
1 on eggs and on
6-transfected cells, our goal was to compare the ability of
mouse eggs to bind fertilin
and laminin. For this purpose
we chose to use fluorescent beads coated with either fertilin
or laminin.
We first had to establish a method to solubilize fertilin from mature (fertilization-competent) sperm using a nondenaturing detergent. Fertilin
(the ~57-kD form) from
mature fertilization-competent sperm, harvested from the
cauda epididymis, is highly resistant to solubilization with
nonionic detergents (Fig. 4 a, lane 2; Huovila, A., E. Almeida, and J. White, unpublished data), while proteolytically processed fertilin
(and its larger precursors) from immature sperm, harvested from the testis, the caput
epididymis (Fig. 4 a, lane 1) or to a lesser extent, the corpus epididymis, are readily solubilized in a variety of nonionic detergents (Huovila, A., E. Almeida, M. Chen, and J. White, unpublished data). Recent evidence suggests that
there are alternate forms of fertilin
on mouse sperm
(Huovila, A., I. Kärkkäinen, C. Rea, and J. White, unpublished data). The cytoplasmic tail antibody used in this paper specifically recognizes the ~57-kD form. Proteolytically processed fertilin
(~57 kD) from mature cauda
epididymal sperm was not solubilized in our lysis buffer
containing the zwitterionic detergent, CHAPS (Fig. 4 a,
lane 2). However, inclusion of gelsolin, an actin severing
protein, permitted us to extract fertilin
(~57 kD) in the
CHAPS-containing lysis buffer (Fig. 4 a, lane 3). Fertilin
(~57 kD) from mature sperm, solubilized in CHAPS/ gelsolin (Fig. 4 a, lane 3), comigrated on a Western blot
(~57 kD) with fertilin from caput epididymal sperm solubilized in the absence of gelsolin (Fig. 4 a, lane 1). The fact
that gelsolin is required to solubilize fertilin
(~57 kD)
from mature, but not from immature, sperm suggests that
it is somehow associated with the actin cytoskeleton of
mature sperm. These observations may be related to the
developmentally regulated posterior head localization of
fertilin
in mature fertilization-competent sperm (Blobel
et al., 1990
; Phelps et al., 1990
; Hunnicutt et al., 1997
).
|
We next assessed the ability of a fertilin cytoplasmic
tail antibody to immunoprecipitate fertilin
from samples
of cell-surface biotinylated sperm solubilized in CHAPS/
gelsolin. The antibody precipitated a biotinylated protein
of ~57 kD (Fig. 4 b, lanes 1 and 3) that comigrated with a
band recognized by the same antibody on a Western blot
of nonbiotinylated sperm (Fig. 4 b, lane 7). This band was
not immunoprecipitated with a control antibody against the cytoplasmic tail of the Avian Leukosis and Sarcoma
Virus envelope glycoprotein, anti-ENV (Fig. 4 b, lanes 2 and 4). It was also not detected in lanes containing anti-
fertilin
or anti-ENV antibodies alone (Fig. 4 b, lanes 5 and 6). When the fertilin
and control immunoprecipitates were washed with PBS (Fig. 4 b, lanes 1 and 2) as opposed to the SDS-containing buffer, RIPA (Fig. 4 b, lanes
3 and 4), an additional band was detected (~37 kD), but it
was not specific to the fertilin
immunoprecipitation. These findings suggest that fertilin
is the major sperm
surface protein immunoprecipitated with our anti-fertilin
cytoplasmic tail antibody.
We asked whether fluorescent beads coated with fertilin
could bind specifically to eggs. To do this, fluorescent
latex beads were coated with affinity-purified fertilin
-cytoplasmic tail antibody and incubated with a CHAPS/
gelsolin lysate from mature capacitated mouse sperm. After washing, the beads were incubated with zona-free
mouse eggs. As seen in Fig. 5 (left), fertilin
-coated beads
bound to eggs. If the fluorescent beads coated with the fertilin
-cytoplasmic tail antibody were incubated in a somatic cell lysate instead of a sperm lysate, only a low level of binding was observed (Fig. 5, middle). If the fluorescent
beads were coated with an irrelevant polyclonal anti-cytoplasmic tail antibody, anti-ENV, and then in a sperm
lysate, only a low level of binding was observed (Fig. 5,
right). These data indicate that fertilin
-coated beads
bind to zona-free mouse eggs and that this binding depends
on fertilin
.
|
We next explored the molecular basis for the fertilin
-bead binding to eggs. As seen in Fig. 6, a fertilin
peptide analogue inhibited binding of fertilin
-coated beads
to eggs (Fig. 6,
) whereas a scrambled fertilin
disintegrin loop peptide analogue did not (Fig. 6,
scr). We next
tested the effects of GoH3, a function-blocking anti-
6
mAb (Sonnenberg et al., 1988
) and J1B5, a non-function-blocking anti-
6 mAb (Damsky et al., 1992
). As seen in
Fig. 6, GoH3 decreased fertilin bead-binding to eggs (compare GoH3 with Ct). In contrast, J1B5 did not inhibit
binding of fertilin
-coated beads. In fact, J1B5 appeared
to enhance the binding of fertilin-coated beads (Fig. 6,
J1B5). The enhancing effect of J1B5 was seen in replicate
experiments. We have recently observed similar inhibitory
and stimulatory effects of GoH3 and J1B5, respectively, on the binding of fluorescent beads coated with a recombinant fertilin
disintegrin domain expressed in insect cells
(Bigler, D., M.S. Chen, Y. Takahashi, E.A.C. Almeida,
and J.M. White, manuscript in preparation). Collectively,
the results presented in Fig. 6 indicate that binding of fertilin
-coated beads to eggs is mediated, at least in part, by
the disintegrin loop of fertilin
and by the integrin
6 subunit.
|
Inverse Effects of Integrin Avidity/Affinity Modulators on Binding of Fertilin- and Laminin E8-coated Beads to Mouse Eggs
We next assessed the effects of integrin avidity/affinity
modulators on the binding of fertilin- (Figs. 7 and 10) and
laminin E8- (see Figs. 8-10) coated fluorescent beads
to mouse eggs. Because beads coated with whole laminin formed large aggregates in suspension, we used the
elastase digestion fragment, E8, which contains the major
integrin 6
1 binding domain (Goodman, 1992
; Yurchenco and O'Rear, 1994
). We first analyzed fertilin- (Fig.
7) and laminin E8- (see Figs. 8 and 9) bead binding individually. We then analyzed fertilin and laminin E8 bead
binding in a competition experiment (see Fig. 10). As seen
in Fig. 7 a, PMA (middle) and Mn2+ (right) inhibited binding of fertilin
-coated beads to mouse eggs (fertilin-bead
binding in the presence of Mg2+ alone [1 mM] was similar
to that seen in the Ca2+/Mg2+ control media; Almeida, E.,
M. Chen, and J. White, unpublished data). As seen in Fig.
7 b, the effect of PMA on fertilin bead binding was dose-dependent.
|
|
|
|
We next assessed the effects of PMA and Mn2+ on the
ability of fluorescent beads coated with the laminin E8
fragment to bind to mouse eggs. Although mouse eggs express significant levels of 6 on their surface, presumably
in complex with
1 (Tarone et al., 1993
; Almeida et al.,
1995
; Evans et al., 1997
; Chen, M., and J. White, unpublished data), the eggs as we isolated and prepared them (in
a Ca2+-containing medium) showed virtually no affinity
for the laminin E8 beads (Fig. 8 a, left). Treatment with
PMA did not induce binding of laminin E8-coated beads
(Fig. 8 a, middle). However, Mn2+ dramatically increased
binding of laminin E8-coated beads (Fig. 8 a, right). The
stimulation of laminin E8-bead binding first occurred at
~50-100 µM Mn2+ and reached a maximum at 200 µM
Mn2+ (Fig. 8 b). Binding of laminin E8-coated beads to
eggs in the presence of Mn2+ was inhibited by GoH3 (Fig.
9, middle) but not by J1B5 (Fig. 9, right). Fluorescent
beads coated with the laminin elastase digestion fragment
E3, which binds to dystroglycan (Ekblom, 1996
), did not
bind to eggs under any conditions tested (Almeida, E., M. Chen, and J. White, unpublished data).
It has been shown recently that in order for 6 to be
switched into its high binding state (for laminin) by PMA,
but not by Mn2+, it must be cleaved into its two disulfide-bonded subunits (Delwel et al., 1996
). Given the phenotype of laminin E8 binding to eggs (Fig. 8 a), we investigated whether the
6 subunit on the egg is proteolytically
processed. We detected significant levels of proteolytically
processed
6 on the egg surface. The apparent molecular
masses of the two subunits on reducing SDS gels were ~120 and ~30 kD (Chen, M., and J. White, unpublished
data), in good agreement with the sizes reported for the
6
subunit from somatic cells (Delwel et al., 1996
). Therefore,
a lack of proteolytic processing does not appear to be responsible for the absence of PMA responsiveness observed in the laminin E8-egg binding assay. The fact that
PMA induces adhesion of
6-expressing somatic cells to
laminin but does not induce laminin E8 binding to eggs
may be due to either fundamental differences between the
6
1 integrin in eggs versus somatic cells (see Discussion)
or simply due to differences between a laminin adhesion
assay versus a laminin bead-binding assay.
We next directly compared the ability of mouse eggs to
bind fertilin- and laminin E8-coated beads in Ca2+- and
Mn2+-containing egg medium. To do this, a mixture of approximately equal numbers of yellow-green beads coated
with fertilin and crimson beads coated with laminin E8
were incubated with eggs in TE medium, which contains
2.4 mM Ca2+, with or without additional Mn2+. Eggs in
Ca2+-containing medium bound large numbers of fertilin-coated beads (yellow-green) but virtually no laminin E8-
coated beads (crimson) (Fig. 10, left). In striking contrast,
eggs in Mn2+-supplemented egg medium bound large
numbers of laminin E8-coated beads (crimson), and a significantly reduced number of fertilin-coated beads (yellow-green) (Fig. 10, right).
Integrin function and clustering have been shown to be
dependent upon cytoskeletal linkages (Kucik et al., 1996;
Lub et al., 1997
; Yauch et al., 1997
). Therefore, we examined the effects of latrunculin, an agent that inhibits actin
polymerization and sequesters actin monomers (Spector
et al., 1989
; Lamaze et al., 1997
), on the binding of fertilin
- and laminin E8-coated beads to eggs. Fertilization-competent mouse eggs normally display dense cortical actin, as visualized by phalloidin staining, particularly concentrated over the meiotic spindle (Reima and Lehtonen,
1985
; Longo, 1987
). Treatment of eggs with latrunculin significantly decreased the amount of polymerized actin
stained with phalloidin (Fig. 11, top). Parallel treatment
with latrunculin decreased fertilin
bead binding to eggs
(Fig. 11, middle), but did not increase laminin E8 bead
binding (Fig. 11, bottom).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In previous work we presented evidence that the integrin
6
1, on both eggs and
6-transfected somatic cells, can
interact with the ADAM protein, fertilin
(Almeida et al.,
1995
). Here we present additional evidence for this interaction. Beads coated with fertilin
captured from a sperm
lysate bind to eggs in a manner that is specifically inhibited
by both a fertilin
disintegrin loop peptide analogue as
well as the anti-
6 mAb, GoH3. Analogous studies using
beads coated with fertilin
disintegrin domain-containing constructs made in insect cells (Bigler, D., M.S. Chen, Y. Takahashi, E.A.C. Almeida, and J.M. White, manuscript
in preparation) as well as with an ELISA (Huovila, A.,
and J. White, unpublished data) corroborate this conclusion. In a recent study using a fertilin
construct produced
in bacteria, Evans et al. (1997)
concluded that fertilin
interacts with a
1 integrin on the egg but not with
6
1.
The difference in results may stem from the use of recombinant proteins produced in bacterial (Evans et al., 1997
) versus eukaryotic (Bigler, D., et al., manuscript in preparation) systems coupled with other technical differences.
Since it appears that 6
1 can interact with the ADAM
protein fertilin
as well as with its well-characterized
ECM ligand laminin, we tested the hypothesis that different states of
6
1 interact with fertilin
and laminin. To
do this we compared the effects of agents that modulate
laminin adhesion to
6
1 on the ability of cells transfected
with
6 to adhere to laminin or to bind sperm. We tested
the ability of eggs to bind beads coated with either fertilin
or laminin. Finally, we compared the effect of the actin cytoskeletal-disrupting agent, latrunculin, on fertilin and
laminin bead-binding to eggs.
With 6-transfected cells we found that agents that increase laminin binding (e.g., PMA, Mn2+) inhibit sperm
binding. Conversely, agents that inhibit laminin binding
enhance sperm binding. In our second set of experiments we found that eggs in Ca2+-containing medium bind beads
coated with fertilin, but do not bind beads coated with the
laminin E8 fragment. In Mn2+-containing medium, however, eggs show a decided preference for laminin E8-coated
beads. PMA treatment of eggs inhibited binding of fertilin-coated beads, but did not induce binding of laminin E8-coated beads. Disruption of the egg actin cytoskeleton
with latrunculin also decreased binding of fertilin-coated
beads, but did not induce binding of laminin E8-coated beads.
Three State Model for the Egg Integrin 6
1
Our data support the hypothesis that different states of the
integrin 6
1 support binding of the ADAM protein fertilin
and the ECM protein laminin. Although previous
studies have shown that integrins can be activated from
low to high avidity/affinity states for interacting with particular ligands or antibodies (Faull and Ginsberg, 1995
;
Bazzoni and Hemler, 1998
), and although in the case of
2
1 it has been shown that activation of the integrin increases its binding avidity for ECM ligands (collagen and
laminin) but not for a non-ECM ligand, echovirus (Bergelson et al., 1993
), our findings are the first to suggest that
different states of an integrin prefer a cell surface or an
ECM ligand. The classical resting state of
6
1 supports
binding of fertilin but not laminin. In this respect binding
of the disintegrin domain of fertilin
to the resting state of
6
1 resembles the ability of snake disintegrins to bind to
the off state of the platelet integrin (Gould et al., 1990
;
Williams, 1992
; Niewiarowski et al., 1994
). Conversely, the
activated state of
6
1 on the egg strongly supports laminin binding but discourages fertilin binding.
As outlined above, our data suggest that the egg 6
1
integrin can exist in a state, exemplified by native fertilization-competent eggs, that supports binding of the ADAM
protein fertilin
but not laminin (Fig. 12, left). It can also
exist in a state exemplified by treating eggs with Mn2+ that
strongly prefers binding laminin and disfavors the ADAM (Fig. 12, right). Our data further suggest that
6
1 on the
egg can exist in a third state, exemplified by treating eggs
with phorbol esters or latrunculin, with limited affinity for
either ligand (Fig. 12, middle). It may be that in the unfertilized egg
6
1 is tethered to the cortical actin cytoskeleton and binds the ADAM protein fertilin
(Fig. 12, left).
Release from cytoskeletal anchorage (induced in vitro by
treatment with either latrunculin or PMA; Fig. 12, middle)
may promote lateral diffusion of the integrin (Kucik et al.,
1996
; Yauch et al., 1997
). Increase in the lateral mobility of
6
1 may in turn disfavor binding between
6
1 and fertilin and may be a prerequisite for interaction between
6
1 and laminin. High avidity adhesion of laminin to
6
1 may
require subsequent clustering of
6
1 and/or changes in
its conformation (mimicked in vitro by treatment with
Mn2+; Fig. 12, right). It should be noted that in addition to
binding different states of the
6
1 integrin, fertilin
and
laminin may bind to different sites on the integrin. Approximately 5-10-fold more GoH3 is required to inhibit
binding of fertilin
than to inhibit binding of laminin.
|
Possible Mechanisms for Regulating Different States of
the Egg Integrin 6
1
We are currently considering two possibilities for how the
integrin 6
1 is kept in an on state for fertilin, and in an
off state for laminin in unfertilized eggs. The first is that
the
6 or
1 subunits in the egg may differ from those expressed in somatic cells. Although we have found both the
6A and
6B alternatively spliced cytoplasmic tail isoforms in the egg, at both the mRNA (Almeida et al., 1995
)
and protein (Chen, M., Y. Takahashi, and J. White, unpublished data) levels, and although we have detected significant levels of proteolytically processed
6 on the egg surface, it is possible that the
6 or
1 subunits on the egg represent alternatively spliced forms of their ectodomains
(Delwel et al., 1995
; Ziober et al., 1997
) or may be differently posttranslationally modified. The second, and our
currently favored, possibility is that on the egg the
6
1
integrin is associated with a specific set of integrin-associated proteins that may include both plasma membrane
and cytoplasmic proteins (Shattil et al., 1995
; Berditchevski et al., 1996
; Fenczik et al., 1997
; Wei et al., 1997
). Such
integrin-associated proteins may, in turn, influence how
the
6
1 integrin is associated with the actin cytoskeleton
(Berditchevski et al., 1996
; Hemler, 1998
).
Implications for Fertilization and Zygotic Development
Since we have shown that the integrin 6
1 on the egg is
in an off state for binding laminin and in an on state for
binding fertilin, and we can experimentally induce a switch
in its binding state, it is possible that
6
1 exists in alternate binding states during development; for example in
oocytes or embryos. We are particularly interested in ascertaining whether (and when) postfertilization
6
1 may
switch to a state that disfavors fertilin and favors laminin.
Such a switch (or switches, see Fig. 12) may be relevant to
both the noted block to polyspermy that occurs at the egg
plasma membrane (Horvath et al., 1993
; Maluchnik and
Borsuk, 1994
) as well as to the noted requirement for
6
1
to support endoderm cell outgrowth on laminin at the time
of uterine implantation (Stephens et al., 1993
; Sutherland
et al., 1993
). It is still not clear whether the interaction between fertilin
and
6
1 is required for fertilization in
vivo, and if so, for what stages of gametogenesis and/or
fertilization. For example, in the testes
6
1 is expressed
on Sertoli cells at sites devoid of laminin but adjoining developing spermatids (Salanova et al., 1995
). Interactions between fertilin
or other sperm ADAMs and
6
1 on
Sertoli cells (in an on state for the ADAM) may be involved in Sertoli cell-spermatid interactions. In addition,
ADAM-
6
1 interactions could be involved during the
long journey that sperm make through the male and female reproductive tracts before reaching the egg (Cho et al.,
1998
).
Implications for Other ADAM-Integrin Interactions
Evidence has been presented that the ability of an integrin
to organize a specific ECM ligand binding site can be developmentally regulated (Ramos and DeSimone, 1996;
Martin-Bermudo et al., 1998
). Hence, it seems plausible
that there might be developmental situations, in addition
to fertilization, when cells use differential regulation of an
integrin for binding ECM ligands and cell surface coreceptors, such as ADAMs (Wolfsberg and White, 1996
). For
example, integrin subunits, including
6 and
1, are expressed in developing dermal epithelial cells at sites of
cell-cell contacts (Hertle et al., 1991
) and their coreceptors at these cell contact sites are not known. If their coreceptors are ADAMs, a switch in the affinity of the
6 integrin from a laminin to an ADAM binding mode may be a
key step in the switch from a cell-ECM mode, for binding to the basement membrane, to a cell-cell binding mode,
upon leaving the basement membrane.
Given the large number of different ADAMs and integrins and their widespread distributions, it is plausible that
many more ADAM-integrin interactions exist. The disintegrin domain of ADAM 15 has been reported recently to
interact with the integrin v
3 (Zhang et al., 1998
). It will
be interesting to see whether binding of ADAM 15 and
fibronectin are inversely regulated by avidity/affinity modulators as we have seen here for ADAM 2 and laminin interacting with
6
1. As more ADAM-integrin interactions are identified, it will be interesting to determine
their roles in development and their mechanisms of regulation.
![]() |
Footnotes |
---|
Address correspondence to Dr. Judith M. White, University of Virginia, Department of Cell Biology, Health Sciences Center Box 439, Charlottesville, VA 22908. Tel.: 804-924-2593. Fax: 804-982-3912. E-mail: jw7g{at}virginia.edu
Received for publication 12 August 1998 and in revised form 24 November 1998.
The first two authors contributed equally.
Present addresses: Dr. A.-P.J. Huovila, Inst. Medical Technology,
Univ. Tampere, Tampere, Finland FIN-33101; Dr. E.A.C. Almeida, Dept.
Stomatology, Univ. California, San Francisco, CA 94143-0512.
We thank the following individuals for their generous gifts: Dr. Peter Yurchenco, University of Medicine and Dentistry of New Jersey, for the laminin E8 fragment; Dr. Paul Janmey, Harvard University, for gelsolin; Dr. Caroline Damsky, University of California at San Francisco, for the J1B5 mAb; and the Biomolecular Research Facility at the University of Virginia for synthesis of disintegrin loop peptide analogues. We also thank Drs. Douglas DeSimone and Ann Sutherland for critical suggestions on the manuscript, and Dr. Gene Marcantonio for suggesting that we test a cytoskeletal disrupting agent.
This work was supported by a grant (GM48739) from the National Institutes of Health to J.M. White. A. Huovila was supported in part by a fellowship from the Human Frontiers Science Program Organization. M. Chen was supported in part by a Medical Scientist Training Program grant, and a Cell and Molecular Biology Training grant (University of Virginia). Y. Takahashi was supported by fellowships from the Japanese Society for the Promotion of Science for Young Scientists and from the Lalor Foundation.
![]() |
Abbreviations used in this paper |
---|
ADAM, a disintegrin and metalloprotease; ECM, extracellular matrix; ENV, envelope; TE, embryo culture medium.
![]() |
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