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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 PMA and genistein on  laminin and sperm binding to  α6-transfected P388D1 cells.  (Left) Laminin adhesion and  (right) sperm binding assays  were performed in parallel. (a)  α6B-transfected cells were either untreated (hatched bars) or  treated with the indicated  amount of PMA (black bars).  Mock-transfected P388D1 cells  (gray bars) were either untreated (0) or treated with 100  ng/ml PMA (100). (b) α6B-transfected cells were either  maintained in normal media (no  PMA, no genistein; hatched  bars) or pretreated with 100 ng/ ml PMA and increasing concentrations of genistein (white  bars). Laminin adhesion assays  were performed in quadruplicate. Sperm binding assays were  conducted in duplicate wells,  and ∼500 cells were counted for  each condition. PMA (100 ng/ml) induced a (a, left) 3.2-fold and (b, left) 4.8-fold increase in laminin binding, and a (a, right) 6-fold and  (b, right) 4.6-fold decrease in sperm binding, respectively. Values indicate average ± standard error (SE).
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Figure 1: Comparison of the effects of PMA and genistein on laminin and sperm binding to α6-transfected P388D1 cells. (Left) Laminin adhesion and (right) sperm binding assays were performed in parallel. (a) α6B-transfected cells were either untreated (hatched bars) or treated with the indicated amount of PMA (black bars). Mock-transfected P388D1 cells (gray bars) were either untreated (0) or treated with 100 ng/ml PMA (100). (b) α6B-transfected cells were either maintained in normal media (no PMA, no genistein; hatched bars) or pretreated with 100 ng/ ml PMA and increasing concentrations of genistein (white bars). Laminin adhesion assays were performed in quadruplicate. Sperm binding assays were conducted in duplicate wells, and ∼500 cells were counted for each condition. PMA (100 ng/ml) induced a (a, left) 3.2-fold and (b, left) 4.8-fold increase in laminin binding, and a (a, right) 6-fold and (b, right) 4.6-fold decrease in sperm binding, respectively. Values indicate average ± standard error (SE).

Mentions: When transfected with the α6 subunit cDNA P388D1 mouse macrophages express α6 in complex with a protein the size of the β1 integrin subunit on the cell surface. No protein the size of the β4 subunit, the only other known α6 binding partner (Sonnenberg et al., 1987; Hemler et al., 1989), was seen (Shaw et al., 1993). As shown previously, (Shaw et al., 1993) and in Fig. 1 a, left, α6-transfected macrophages adhered to laminin to a greater extent than their mock-transfected counterparts. This adhesion could be stimulated by treatment with PMA. As shown previously (Almeida et al., 1995) and in Fig. 1 a, right, α6-transfected macrophages bound more sperm than their mock-transfected counterparts. However, in contrast to its enhancing effect on laminin adhesion, PMA inhibited sperm binding to parallel cultures of α6-transfected macrophages (Fig. 1 a, right). As observed previously (Shaw, L., and A. Mercurio, unpublished data), genistein, a tyrosine kinase inhibitor, reversed the PMA stimulation of laminin adhesion to α6-transfected macrophages (Fig. 1 b, left). In contrast, genistein restored sperm binding to parallel cultures of PMA-treated α6-transfected macrophages (Fig. 1 b, right).


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 PMA and genistein on  laminin and sperm binding to  α6-transfected P388D1 cells.  (Left) Laminin adhesion and  (right) sperm binding assays  were performed in parallel. (a)  α6B-transfected cells were either untreated (hatched bars) or  treated with the indicated  amount of PMA (black bars).  Mock-transfected P388D1 cells  (gray bars) were either untreated (0) or treated with 100  ng/ml PMA (100). (b) α6B-transfected cells were either  maintained in normal media (no  PMA, no genistein; hatched  bars) or pretreated with 100 ng/ ml PMA and increasing concentrations of genistein (white  bars). Laminin adhesion assays  were performed in quadruplicate. Sperm binding assays were  conducted in duplicate wells,  and ∼500 cells were counted for  each condition. PMA (100 ng/ml) induced a (a, left) 3.2-fold and (b, left) 4.8-fold increase in laminin binding, and a (a, right) 6-fold and  (b, right) 4.6-fold decrease in sperm binding, respectively. Values indicate average ± standard error (SE).
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Figure 1: Comparison of the effects of PMA and genistein on laminin and sperm binding to α6-transfected P388D1 cells. (Left) Laminin adhesion and (right) sperm binding assays were performed in parallel. (a) α6B-transfected cells were either untreated (hatched bars) or treated with the indicated amount of PMA (black bars). Mock-transfected P388D1 cells (gray bars) were either untreated (0) or treated with 100 ng/ml PMA (100). (b) α6B-transfected cells were either maintained in normal media (no PMA, no genistein; hatched bars) or pretreated with 100 ng/ ml PMA and increasing concentrations of genistein (white bars). Laminin adhesion assays were performed in quadruplicate. Sperm binding assays were conducted in duplicate wells, and ∼500 cells were counted for each condition. PMA (100 ng/ml) induced a (a, left) 3.2-fold and (b, left) 4.8-fold increase in laminin binding, and a (a, right) 6-fold and (b, right) 4.6-fold decrease in sperm binding, respectively. Values indicate average ± standard error (SE).
Mentions: When transfected with the α6 subunit cDNA P388D1 mouse macrophages express α6 in complex with a protein the size of the β1 integrin subunit on the cell surface. No protein the size of the β4 subunit, the only other known α6 binding partner (Sonnenberg et al., 1987; Hemler et al., 1989), was seen (Shaw et al., 1993). As shown previously, (Shaw et al., 1993) and in Fig. 1 a, left, α6-transfected macrophages adhered to laminin to a greater extent than their mock-transfected counterparts. This adhesion could be stimulated by treatment with PMA. As shown previously (Almeida et al., 1995) and in Fig. 1 a, right, α6-transfected macrophages bound more sperm than their mock-transfected counterparts. However, in contrast to its enhancing effect on laminin adhesion, PMA inhibited sperm binding to parallel cultures of α6-transfected macrophages (Fig. 1 a, right). As observed previously (Shaw, L., and A. Mercurio, unpublished data), genistein, a tyrosine kinase inhibitor, reversed the PMA stimulation of laminin adhesion to α6-transfected macrophages (Fig. 1 b, left). In contrast, genistein restored sperm binding to parallel cultures of PMA-treated α6-transfected macrophages (Fig. 1 b, right).

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