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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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Comparison of the effects of Mn2+ and Ca2+ on laminin and sperm binding to α6-transfected P388D1 cells. (Left)  Laminin adhesion and (right)  sperm binding assays were performed in parallel on α6B-transfected P388D1 cells. (a) (Open  bars) Cells were incubated in  Puck's saline A supplemented with  increasing concentrations of MnCl2.  (b) (Gray bars) Cells were incubated in Puck's saline A containing 0.5 mM MnCl2 and increasing concentrations of CaCl2.  Binding in Puck's saline A containing 1.8 mM CaCl2 and 0.8 mM  MgCl2 in the absence (hatched  bars; α6B, Ca2+/Mg2+) and presence (black bars; α6B, Ca2+/ Mg2+, 100 ng/ml PMA) of 100  ng/ml PMA are shown for comparison. Data analyses are as described in the legend to Fig. 1.
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Figure 2: Comparison of the effects of Mn2+ and Ca2+ on laminin and sperm binding to α6-transfected P388D1 cells. (Left) Laminin adhesion and (right) sperm binding assays were performed in parallel on α6B-transfected P388D1 cells. (a) (Open bars) Cells were incubated in Puck's saline A supplemented with increasing concentrations of MnCl2. (b) (Gray bars) Cells were incubated in Puck's saline A containing 0.5 mM MnCl2 and increasing concentrations of CaCl2. Binding in Puck's saline A containing 1.8 mM CaCl2 and 0.8 mM MgCl2 in the absence (hatched bars; α6B, Ca2+/Mg2+) and presence (black bars; α6B, Ca2+/ Mg2+, 100 ng/ml PMA) of 100 ng/ml PMA are shown for comparison. Data analyses are as described in the legend to Fig. 1.

Mentions: 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 α6-transfected macrophages to laminin. In contrast, Mn2+ inhibited sperm binding to α6-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 α6-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 α6-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).


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)

Comparison of the effects of Mn2+ and Ca2+ on laminin and sperm binding to α6-transfected P388D1 cells. (Left)  Laminin adhesion and (right)  sperm binding assays were performed in parallel on α6B-transfected P388D1 cells. (a) (Open  bars) Cells were incubated in  Puck's saline A supplemented with  increasing concentrations of MnCl2.  (b) (Gray bars) Cells were incubated in Puck's saline A containing 0.5 mM MnCl2 and increasing concentrations of CaCl2.  Binding in Puck's saline A containing 1.8 mM CaCl2 and 0.8 mM  MgCl2 in the absence (hatched  bars; α6B, Ca2+/Mg2+) and presence (black bars; α6B, Ca2+/ Mg2+, 100 ng/ml PMA) of 100  ng/ml PMA are shown for comparison. Data analyses are as described in the legend to Fig. 1.
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getmorefigures.php?uid=PMC2132920&req=5

Figure 2: Comparison of the effects of Mn2+ and Ca2+ on laminin and sperm binding to α6-transfected P388D1 cells. (Left) Laminin adhesion and (right) sperm binding assays were performed in parallel on α6B-transfected P388D1 cells. (a) (Open bars) Cells were incubated in Puck's saline A supplemented with increasing concentrations of MnCl2. (b) (Gray bars) Cells were incubated in Puck's saline A containing 0.5 mM MnCl2 and increasing concentrations of CaCl2. Binding in Puck's saline A containing 1.8 mM CaCl2 and 0.8 mM MgCl2 in the absence (hatched bars; α6B, Ca2+/Mg2+) and presence (black bars; α6B, Ca2+/ Mg2+, 100 ng/ml PMA) of 100 ng/ml PMA are shown for comparison. Data analyses are as described in the legend to Fig. 1.
Mentions: 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 α6-transfected macrophages to laminin. In contrast, Mn2+ inhibited sperm binding to α6-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 α6-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 α6-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).

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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Related in: MedlinePlus