Limits...
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).

Show MeSH

Related in: MedlinePlus

Effects of PMA and  Mn2+ on binding of fertilin-coated beads to zona-free eggs.  Eggs were collected in M199  medium and treated with acidic  Tyrode's solution to remove the  zonae, and allowed to recover  for 3 h at 37°C in M199 as described in Materials and Methods. (a) Eggs were placed in  Puck's saline A containing either 1.8 mM CaCl2 and 0.8 mM  MgCl2 (Ct), 1.8 mM CaCl2, 0.8  mM MgCl2 and 50 ng/ml PMA  (PMA), or 1 mM MnCl2 (Mn2+).  For the PMA samples, eggs  were pretreated with PMA for  10 min. Eggs were assayed for  binding of fertilin-coated beads  as described in the legend to Fig.  5. (b) Eggs were placed in  Puck's saline A containing 1.8  mM CaCl2, 0.8 mM MgCl2, and  the indicated concentration of  PMA. Eggs were pretreated  with PMA for 10 min and assayed for the binding of fertilin-coated beads as described  above. Paired fluorescence and  phase-contrast images from representative experiments are  shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132920&req=5

Figure 7: Effects of PMA and Mn2+ on binding of fertilin-coated beads to zona-free eggs. Eggs were collected in M199 medium and treated with acidic Tyrode's solution to remove the zonae, and allowed to recover for 3 h at 37°C in M199 as described in Materials and Methods. (a) Eggs were placed in Puck's saline A containing either 1.8 mM CaCl2 and 0.8 mM MgCl2 (Ct), 1.8 mM CaCl2, 0.8 mM MgCl2 and 50 ng/ml PMA (PMA), or 1 mM MnCl2 (Mn2+). For the PMA samples, eggs were pretreated with PMA for 10 min. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. (b) Eggs were placed in Puck's saline A containing 1.8 mM CaCl2, 0.8 mM MgCl2, and the indicated concentration of PMA. Eggs were pretreated with PMA for 10 min and assayed for the binding of fertilin-coated beads as described above. Paired fluorescence and phase-contrast images from representative experiments are shown.

Mentions: 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 α6β1 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 β–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.


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 and  Mn2+ on binding of fertilin-coated beads to zona-free eggs.  Eggs were collected in M199  medium and treated with acidic  Tyrode's solution to remove the  zonae, and allowed to recover  for 3 h at 37°C in M199 as described in Materials and Methods. (a) Eggs were placed in  Puck's saline A containing either 1.8 mM CaCl2 and 0.8 mM  MgCl2 (Ct), 1.8 mM CaCl2, 0.8  mM MgCl2 and 50 ng/ml PMA  (PMA), or 1 mM MnCl2 (Mn2+).  For the PMA samples, eggs  were pretreated with PMA for  10 min. Eggs were assayed for  binding of fertilin-coated beads  as described in the legend to Fig.  5. (b) Eggs were placed in  Puck's saline A containing 1.8  mM CaCl2, 0.8 mM MgCl2, and  the indicated concentration of  PMA. Eggs were pretreated  with PMA for 10 min and assayed for the binding of fertilin-coated beads as described  above. Paired fluorescence and  phase-contrast images from representative experiments are  shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Effects of PMA and Mn2+ on binding of fertilin-coated beads to zona-free eggs. Eggs were collected in M199 medium and treated with acidic Tyrode's solution to remove the zonae, and allowed to recover for 3 h at 37°C in M199 as described in Materials and Methods. (a) Eggs were placed in Puck's saline A containing either 1.8 mM CaCl2 and 0.8 mM MgCl2 (Ct), 1.8 mM CaCl2, 0.8 mM MgCl2 and 50 ng/ml PMA (PMA), or 1 mM MnCl2 (Mn2+). For the PMA samples, eggs were pretreated with PMA for 10 min. Eggs were assayed for binding of fertilin-coated beads as described in the legend to Fig. 5. (b) Eggs were placed in Puck's saline A containing 1.8 mM CaCl2, 0.8 mM MgCl2, and the indicated concentration of PMA. Eggs were pretreated with PMA for 10 min and assayed for the binding of fertilin-coated beads as described above. Paired fluorescence and phase-contrast images from representative experiments are shown.
Mentions: 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 α6β1 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 β–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.

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