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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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Effects of PMA, Mn2+, and Ca2+ on sperm binding to  zona-free eggs. (a and b) Eggs were placed in drops of TE (that  contains 2.4 mM CaCl2 and 0.47 mM MgCl2), containing increasing concentrations of (a) PMA or (b) MnCl2 for 15 min, and incubated with capacitated sperm. (c) Eggs were pretreated in drops  of Puck's saline A supplemented with increasing concentrations  of CaCl2, and incubated with capacitated sperm. After 1 h at  37°C, eggs were washed, fixed, and analyzed for sperm binding by  phase-contrast microscopy. Approximately 30 eggs were used per  condition. Values indicate average ± SE.
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Figure 3: Effects of PMA, Mn2+, and Ca2+ on sperm binding to zona-free eggs. (a and b) Eggs were placed in drops of TE (that contains 2.4 mM CaCl2 and 0.47 mM MgCl2), containing increasing concentrations of (a) PMA or (b) MnCl2 for 15 min, and incubated with capacitated sperm. (c) Eggs were pretreated in drops of Puck's saline A supplemented with increasing concentrations of CaCl2, and incubated with capacitated sperm. After 1 h at 37°C, eggs were washed, fixed, and analyzed for sperm binding by phase-contrast microscopy. Approximately 30 eggs were used per condition. Values indicate average ± SE.

Mentions: 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 α6-expressing cells (Fig. 1 b, right), the eggs lysed.


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)

Effects of PMA, Mn2+, and Ca2+ on sperm binding to  zona-free eggs. (a and b) Eggs were placed in drops of TE (that  contains 2.4 mM CaCl2 and 0.47 mM MgCl2), containing increasing concentrations of (a) PMA or (b) MnCl2 for 15 min, and incubated with capacitated sperm. (c) Eggs were pretreated in drops  of Puck's saline A supplemented with increasing concentrations  of CaCl2, and incubated with capacitated sperm. After 1 h at  37°C, eggs were washed, fixed, and analyzed for sperm binding by  phase-contrast microscopy. Approximately 30 eggs were used per  condition. Values indicate average ± SE.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effects of PMA, Mn2+, and Ca2+ on sperm binding to zona-free eggs. (a and b) Eggs were placed in drops of TE (that contains 2.4 mM CaCl2 and 0.47 mM MgCl2), containing increasing concentrations of (a) PMA or (b) MnCl2 for 15 min, and incubated with capacitated sperm. (c) Eggs were pretreated in drops of Puck's saline A supplemented with increasing concentrations of CaCl2, and incubated with capacitated sperm. After 1 h at 37°C, eggs were washed, fixed, and analyzed for sperm binding by phase-contrast microscopy. Approximately 30 eggs were used per condition. Values indicate average ± SE.
Mentions: 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 α6-expressing cells (Fig. 1 b, right), the eggs lysed.

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