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Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Chen MS, Almeida EA, Huovila AP, Takahashi Y, Shaw LM, Mercurio AM, White JM - J. Cell Biol. (1999)

Bottom Line: 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.Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-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 beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 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 (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

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Binding of fertilin-coated beads to zona-free eggs. Yellow-green fluorescent latex beads were coated with an antibody  (AB) against the fertilin β–cytoplasmic tail (anti-fertilin) or with  a control antibody (anti-ENV). Beads were then incubated in either a sperm (sperm) or a macrophage (cell) lysate (LYS.). After  the beads were quenched (with anti–rabbit IgG), washed, and  sonicated, they were incubated with eggs for 1 h in TE as described in Materials and Methods. The eggs were washed and analyzed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown.
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Figure 5: Binding of fertilin-coated beads to zona-free eggs. Yellow-green fluorescent latex beads were coated with an antibody (AB) against the fertilin β–cytoplasmic tail (anti-fertilin) or with a control antibody (anti-ENV). Beads were then incubated in either a sperm (sperm) or a macrophage (cell) lysate (LYS.). After the beads were quenched (with anti–rabbit IgG), washed, and sonicated, they were incubated with eggs for 1 h in TE as described in Materials and Methods. The eggs were washed and analyzed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown.

Mentions: We asked whether fluorescent beads coated with fertilin β could bind specifically to eggs. To do this, fluorescent latex beads were coated with affinity-purified fertilin β–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 β–coated beads bound to eggs. If the fluorescent beads coated with the fertilin β–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 β–coated beads bind to zona-free mouse eggs and that this binding depends on fertilin β.


Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Chen MS, Almeida EA, Huovila AP, Takahashi Y, Shaw LM, Mercurio AM, White JM - J. Cell Biol. (1999)

Binding of fertilin-coated beads to zona-free eggs. Yellow-green fluorescent latex beads were coated with an antibody  (AB) against the fertilin β–cytoplasmic tail (anti-fertilin) or with  a control antibody (anti-ENV). Beads were then incubated in either a sperm (sperm) or a macrophage (cell) lysate (LYS.). After  the beads were quenched (with anti–rabbit IgG), washed, and  sonicated, they were incubated with eggs for 1 h in TE as described in Materials and Methods. The eggs were washed and analyzed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132920&req=5

Figure 5: Binding of fertilin-coated beads to zona-free eggs. Yellow-green fluorescent latex beads were coated with an antibody (AB) against the fertilin β–cytoplasmic tail (anti-fertilin) or with a control antibody (anti-ENV). Beads were then incubated in either a sperm (sperm) or a macrophage (cell) lysate (LYS.). After the beads were quenched (with anti–rabbit IgG), washed, and sonicated, they were incubated with eggs for 1 h in TE as described in Materials and Methods. The eggs were washed and analyzed by confocal microscopy. Paired fluorescence and phase-contrast images from a representative experiment are shown.
Mentions: We asked whether fluorescent beads coated with fertilin β could bind specifically to eggs. To do this, fluorescent latex beads were coated with affinity-purified fertilin β–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 β–coated beads bound to eggs. If the fluorescent beads coated with the fertilin β–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 β–coated beads bind to zona-free mouse eggs and that this binding depends on fertilin β.

Bottom Line: 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.Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-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 beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 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 (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

Show MeSH
Related in: MedlinePlus