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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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Solubilization and immunoprecipitation of fertilin β.  (a) Sperm from the caput (lane 1) or cauda (lanes 2 and 3) epididymis were recovered and solubilized in CHAPS lysis buffer in  the absence (lane 1 and 2) or presence (lane 3) of gelsolin. Solubilized proteins were precipitated with ConA as described in  Materials and Methods and boiled in 2× SDS gel sample buffer.  Samples were subjected to 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates  the major fertilin β band recognized on mature sperm (mol.  mass = ∼57 kD). The higher molecular mass band seen in lane 1  (mol. mass = ∼100 kD) represents the full-length fertilin β precursor. − indicates blank lanes. (b) Biotinylated sperm from the  cauda epididymis were solubilized in CHAPS/gelsolin, as described in Materials and Methods. Cleared lysates were immunoprecipitated with protein A agarose beads precoupled with an  antibody against the fertilin β cytoplasmic tail (lanes 1 and 3) or a  control antibody, anti-ENV (lanes 2 and 4), for 1 h at 4°C. Samples were then divided in half and washed either twice with PBS  (lanes 1 and 2) or seven times with RIPA buffer (lanes 3 and 4). 1  μg anti–fertilin β (lane 5) or anti-ENV (lane 6) IgGs were precoupled to 5 μl protein A agarose beads, and the beads were  washed twice with PBS. All samples were subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with Vectastain Elite ABC reagent as described  in Materials and Methods. For the sample shown in lane 7, sperm  from the cauda epididymis were solubilized by direct boiling in  2× SDS gel sample buffer, subjected to 10% SDS-PAGE under  nonreducing conditions, transferred to nitrocellulose, and blotted  with an antibody to the cytoplasmic tail of fertilin β. The arrow  indicates the fertilin β band which corresponds to the ∼57-kD  mature fertilin lacking the prodomain and the metalloprotease  domain.
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Figure 4: Solubilization and immunoprecipitation of fertilin β. (a) Sperm from the caput (lane 1) or cauda (lanes 2 and 3) epididymis were recovered and solubilized in CHAPS lysis buffer in the absence (lane 1 and 2) or presence (lane 3) of gelsolin. Solubilized proteins were precipitated with ConA as described in Materials and Methods and boiled in 2× SDS gel sample buffer. Samples were subjected to 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates the major fertilin β band recognized on mature sperm (mol. mass = ∼57 kD). The higher molecular mass band seen in lane 1 (mol. mass = ∼100 kD) represents the full-length fertilin β precursor. − indicates blank lanes. (b) Biotinylated sperm from the cauda epididymis were solubilized in CHAPS/gelsolin, as described in Materials and Methods. Cleared lysates were immunoprecipitated with protein A agarose beads precoupled with an antibody against the fertilin β cytoplasmic tail (lanes 1 and 3) or a control antibody, anti-ENV (lanes 2 and 4), for 1 h at 4°C. Samples were then divided in half and washed either twice with PBS (lanes 1 and 2) or seven times with RIPA buffer (lanes 3 and 4). 1 μg anti–fertilin β (lane 5) or anti-ENV (lane 6) IgGs were precoupled to 5 μl protein A agarose beads, and the beads were washed twice with PBS. All samples were subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with Vectastain Elite ABC reagent as described in Materials and Methods. For the sample shown in lane 7, sperm from the cauda epididymis were solubilized by direct boiling in 2× SDS gel sample buffer, subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates the fertilin β band which corresponds to the ∼57-kD mature fertilin lacking the prodomain and the metalloprotease domain.

Mentions: We first had to establish a method to solubilize fertilin β from mature (fertilization-competent) sperm using a nondenaturing detergent. Fertilin β (the ∼57-kD form) from mature fertilization-competent sperm, harvested from the cauda epididymis, is highly resistant to solubilization with nonionic detergents (Fig. 4 a, lane 2; Huovila, A., E. Almeida, and J. White, unpublished data), while proteolytically processed fertilin β (and its larger precursors) from immature sperm, harvested from the testis, the caput epididymis (Fig. 4 a, lane 1) or to a lesser extent, the corpus epididymis, are readily solubilized in a variety of nonionic detergents (Huovila, A., E. Almeida, M. Chen, and J. White, unpublished data). Recent evidence suggests that there are alternate forms of fertilin β on mouse sperm (Huovila, A., I. Kärkkäinen, C. Rea, and J. White, unpublished data). The cytoplasmic tail antibody used in this paper specifically recognizes the ∼57-kD form. Proteolytically processed fertilin β (∼57 kD) from mature cauda epididymal sperm was not solubilized in our lysis buffer containing the zwitterionic detergent, CHAPS (Fig. 4 a, lane 2). However, inclusion of gelsolin, an actin severing protein, permitted us to extract fertilin β (∼57 kD) in the CHAPS-containing lysis buffer (Fig. 4 a, lane 3). Fertilin β (∼57 kD) from mature sperm, solubilized in CHAPS/ gelsolin (Fig. 4 a, lane 3), comigrated on a Western blot (∼57 kD) with fertilin from caput epididymal sperm solubilized in the absence of gelsolin (Fig. 4 a, lane 1). The fact that gelsolin is required to solubilize fertilin β (∼57 kD) from mature, but not from immature, sperm suggests that it is somehow associated with the actin cytoskeleton of mature sperm. These observations may be related to the developmentally regulated posterior head localization of fertilin β in mature fertilization-competent sperm (Blobel et al., 1990; Phelps et al., 1990; Hunnicutt et al., 1997).


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)

Solubilization and immunoprecipitation of fertilin β.  (a) Sperm from the caput (lane 1) or cauda (lanes 2 and 3) epididymis were recovered and solubilized in CHAPS lysis buffer in  the absence (lane 1 and 2) or presence (lane 3) of gelsolin. Solubilized proteins were precipitated with ConA as described in  Materials and Methods and boiled in 2× SDS gel sample buffer.  Samples were subjected to 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates  the major fertilin β band recognized on mature sperm (mol.  mass = ∼57 kD). The higher molecular mass band seen in lane 1  (mol. mass = ∼100 kD) represents the full-length fertilin β precursor. − indicates blank lanes. (b) Biotinylated sperm from the  cauda epididymis were solubilized in CHAPS/gelsolin, as described in Materials and Methods. Cleared lysates were immunoprecipitated with protein A agarose beads precoupled with an  antibody against the fertilin β cytoplasmic tail (lanes 1 and 3) or a  control antibody, anti-ENV (lanes 2 and 4), for 1 h at 4°C. Samples were then divided in half and washed either twice with PBS  (lanes 1 and 2) or seven times with RIPA buffer (lanes 3 and 4). 1  μg anti–fertilin β (lane 5) or anti-ENV (lane 6) IgGs were precoupled to 5 μl protein A agarose beads, and the beads were  washed twice with PBS. All samples were subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with Vectastain Elite ABC reagent as described  in Materials and Methods. For the sample shown in lane 7, sperm  from the cauda epididymis were solubilized by direct boiling in  2× SDS gel sample buffer, subjected to 10% SDS-PAGE under  nonreducing conditions, transferred to nitrocellulose, and blotted  with an antibody to the cytoplasmic tail of fertilin β. The arrow  indicates the fertilin β band which corresponds to the ∼57-kD  mature fertilin lacking the prodomain and the metalloprotease  domain.
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Related In: Results  -  Collection

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Figure 4: Solubilization and immunoprecipitation of fertilin β. (a) Sperm from the caput (lane 1) or cauda (lanes 2 and 3) epididymis were recovered and solubilized in CHAPS lysis buffer in the absence (lane 1 and 2) or presence (lane 3) of gelsolin. Solubilized proteins were precipitated with ConA as described in Materials and Methods and boiled in 2× SDS gel sample buffer. Samples were subjected to 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates the major fertilin β band recognized on mature sperm (mol. mass = ∼57 kD). The higher molecular mass band seen in lane 1 (mol. mass = ∼100 kD) represents the full-length fertilin β precursor. − indicates blank lanes. (b) Biotinylated sperm from the cauda epididymis were solubilized in CHAPS/gelsolin, as described in Materials and Methods. Cleared lysates were immunoprecipitated with protein A agarose beads precoupled with an antibody against the fertilin β cytoplasmic tail (lanes 1 and 3) or a control antibody, anti-ENV (lanes 2 and 4), for 1 h at 4°C. Samples were then divided in half and washed either twice with PBS (lanes 1 and 2) or seven times with RIPA buffer (lanes 3 and 4). 1 μg anti–fertilin β (lane 5) or anti-ENV (lane 6) IgGs were precoupled to 5 μl protein A agarose beads, and the beads were washed twice with PBS. All samples were subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with Vectastain Elite ABC reagent as described in Materials and Methods. For the sample shown in lane 7, sperm from the cauda epididymis were solubilized by direct boiling in 2× SDS gel sample buffer, subjected to 10% SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and blotted with an antibody to the cytoplasmic tail of fertilin β. The arrow indicates the fertilin β band which corresponds to the ∼57-kD mature fertilin lacking the prodomain and the metalloprotease domain.
Mentions: We first had to establish a method to solubilize fertilin β from mature (fertilization-competent) sperm using a nondenaturing detergent. Fertilin β (the ∼57-kD form) from mature fertilization-competent sperm, harvested from the cauda epididymis, is highly resistant to solubilization with nonionic detergents (Fig. 4 a, lane 2; Huovila, A., E. Almeida, and J. White, unpublished data), while proteolytically processed fertilin β (and its larger precursors) from immature sperm, harvested from the testis, the caput epididymis (Fig. 4 a, lane 1) or to a lesser extent, the corpus epididymis, are readily solubilized in a variety of nonionic detergents (Huovila, A., E. Almeida, M. Chen, and J. White, unpublished data). Recent evidence suggests that there are alternate forms of fertilin β on mouse sperm (Huovila, A., I. Kärkkäinen, C. Rea, and J. White, unpublished data). The cytoplasmic tail antibody used in this paper specifically recognizes the ∼57-kD form. Proteolytically processed fertilin β (∼57 kD) from mature cauda epididymal sperm was not solubilized in our lysis buffer containing the zwitterionic detergent, CHAPS (Fig. 4 a, lane 2). However, inclusion of gelsolin, an actin severing protein, permitted us to extract fertilin β (∼57 kD) in the CHAPS-containing lysis buffer (Fig. 4 a, lane 3). Fertilin β (∼57 kD) from mature sperm, solubilized in CHAPS/ gelsolin (Fig. 4 a, lane 3), comigrated on a Western blot (∼57 kD) with fertilin from caput epididymal sperm solubilized in the absence of gelsolin (Fig. 4 a, lane 1). The fact that gelsolin is required to solubilize fertilin β (∼57 kD) from mature, but not from immature, sperm suggests that it is somehow associated with the actin cytoskeleton of mature sperm. These observations may be related to the developmentally regulated posterior head localization of fertilin β in mature fertilization-competent sperm (Blobel et al., 1990; Phelps et al., 1990; Hunnicutt et al., 1997).

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