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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 fertilin β  peptide analogues and anti-α6  antibodies on binding of fertilin-coated beads to zona-free  eggs. Eggs in TE were either  untreated (Ct) or preincubated  with 250 μM freshly dissolved  peptide analogue (14 residues)  corresponding to either the  predicted binding domain of  fertilin β (β) or a scrambled  fertilin β peptide (βscr). In  other samples, eggs were preincubated with 100 μg/ml of  either a function-blocking  (GoH3) or a non–function-blocking (J1B5) anti-α6 mAb. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. Paired fluorescence and phase-contrast images from a representative experiment are shown.
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Figure 6: Effects of fertilin β peptide analogues and anti-α6 antibodies on binding of fertilin-coated beads to zona-free eggs. Eggs in TE were either untreated (Ct) or preincubated with 250 μM freshly dissolved peptide analogue (14 residues) corresponding to either the predicted binding domain of fertilin β (β) or a scrambled fertilin β peptide (βscr). In other samples, eggs were preincubated with 100 μg/ml of either a function-blocking (GoH3) or a non–function-blocking (J1B5) anti-α6 mAb. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. Paired fluorescence and phase-contrast images from a representative experiment are shown.

Mentions: We next explored the molecular basis for the fertilin β–bead binding to eggs. As seen in Fig. 6, a fertilin β peptide analogue inhibited binding of fertilin β–coated beads to eggs (Fig. 6, β) whereas a scrambled fertilin β disintegrin loop peptide analogue did not (Fig. 6, βscr). We next tested the effects of GoH3, a function-blocking anti-α6 mAb (Sonnenberg et al., 1988) and J1B5, a non–function-blocking anti-α6 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 β–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 β 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 β–coated beads to eggs is mediated, at least in part, by the disintegrin loop of fertilin β and by the integrin α6 subunit.


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 fertilin β  peptide analogues and anti-α6  antibodies on binding of fertilin-coated beads to zona-free  eggs. Eggs in TE were either  untreated (Ct) or preincubated  with 250 μM freshly dissolved  peptide analogue (14 residues)  corresponding to either the  predicted binding domain of  fertilin β (β) or a scrambled  fertilin β peptide (βscr). In  other samples, eggs were preincubated with 100 μg/ml of  either a function-blocking  (GoH3) or a non–function-blocking (J1B5) anti-α6 mAb. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. Paired fluorescence and phase-contrast images from a representative experiment are shown.
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Related In: Results  -  Collection

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

Figure 6: Effects of fertilin β peptide analogues and anti-α6 antibodies on binding of fertilin-coated beads to zona-free eggs. Eggs in TE were either untreated (Ct) or preincubated with 250 μM freshly dissolved peptide analogue (14 residues) corresponding to either the predicted binding domain of fertilin β (β) or a scrambled fertilin β peptide (βscr). In other samples, eggs were preincubated with 100 μg/ml of either a function-blocking (GoH3) or a non–function-blocking (J1B5) anti-α6 mAb. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. Paired fluorescence and phase-contrast images from a representative experiment are shown.
Mentions: We next explored the molecular basis for the fertilin β–bead binding to eggs. As seen in Fig. 6, a fertilin β peptide analogue inhibited binding of fertilin β–coated beads to eggs (Fig. 6, β) whereas a scrambled fertilin β disintegrin loop peptide analogue did not (Fig. 6, βscr). We next tested the effects of GoH3, a function-blocking anti-α6 mAb (Sonnenberg et al., 1988) and J1B5, a non–function-blocking anti-α6 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 β–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 β 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 β–coated beads to eggs is mediated, at least in part, by the disintegrin loop of fertilin β and by the integrin α6 subunit.

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