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Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.

Troyanovsky B, Levchenko T, Månsson G, Matvijenko O, Holmgren L - J. Cell Biol. (2001)

Bottom Line: Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells.Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility.These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics Research and Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 76 Stockholm, Sweden.

ABSTRACT
Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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Yeast two-hybrid screening for angiostatin-binding proteins in a human placenta cDNA library. (A) Yeast transfected with the angiostatin-Gal4 binding domain and BIG3-Gal4 activation domain grow under growth-restricted conditions (−Leu, −His, −Trp) and in the presence of 50 mM 3-amino-1, 2,4-triazol. No growth was detected in yeast transfected with both the angiostatin-Gal4 binding domain and the Gal4 activation domain, or the Gal4 binding domain and the BIG3-Gal4 activation domain. The p53-Gal4 binding domain and SV40LT-Gal4 activation domain were used as positive controls. (B) The table shows the activity of the nonselectable β-gal marker in yeast cells transfected with angiostatin-Gal4 binding and the BIG3-Gal4 activation domain and controls. (C) Binding of the 421 amino acid BIG3 sequence derived from yeast two-hybrid screening was verified by coprecipitation of GST-BIG3 with angiostatin.
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Figure 1: Yeast two-hybrid screening for angiostatin-binding proteins in a human placenta cDNA library. (A) Yeast transfected with the angiostatin-Gal4 binding domain and BIG3-Gal4 activation domain grow under growth-restricted conditions (−Leu, −His, −Trp) and in the presence of 50 mM 3-amino-1, 2,4-triazol. No growth was detected in yeast transfected with both the angiostatin-Gal4 binding domain and the Gal4 activation domain, or the Gal4 binding domain and the BIG3-Gal4 activation domain. The p53-Gal4 binding domain and SV40LT-Gal4 activation domain were used as positive controls. (B) The table shows the activity of the nonselectable β-gal marker in yeast cells transfected with angiostatin-Gal4 binding and the BIG3-Gal4 activation domain and controls. (C) Binding of the 421 amino acid BIG3 sequence derived from yeast two-hybrid screening was verified by coprecipitation of GST-BIG3 with angiostatin.

Mentions: We generated a Gal4-binding domain fusion protein containing the kringle domains 1–4 of angiostatin. This fusion protein was expressed in yeast and did not activate the His or β-gal reporter genes when expressed alone or in combination with control constructs (Fig. 1A and Fig. B). Approximately 2 × 106 clones from a human term placenta cDNA library were screened with the angiostatin-Gal4 construct. Screening under selective conditions (His−, Leu−, and Trp−) generated 37 positive clones in the CG1945 yeast strain. 7 of the 37 clones displayed high β-gal activity after incubation with ONPG at 30°C for 2 h. The DNA from the colonies that contained high β-gal activity was purified and retransfected into yeast strain Y190. Three of the seven of the colonies retained activity in the new yeast strain. Sequence analysis revealed that these three clones were derived from the same gene, and the yeast-derived sequence was subsequently named BIG3. Binding of the BIG3 peptide to angiostatin was verified by coimmunoprecipitation of angiostatin with recombinant GST-tagged BIG3 domain of angiomotin (Fig. 1 C). Angiostatin binding to BIG3 was visualized with antibodies against kringle domains 1–4 by Western blot.


Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.

Troyanovsky B, Levchenko T, Månsson G, Matvijenko O, Holmgren L - J. Cell Biol. (2001)

Yeast two-hybrid screening for angiostatin-binding proteins in a human placenta cDNA library. (A) Yeast transfected with the angiostatin-Gal4 binding domain and BIG3-Gal4 activation domain grow under growth-restricted conditions (−Leu, −His, −Trp) and in the presence of 50 mM 3-amino-1, 2,4-triazol. No growth was detected in yeast transfected with both the angiostatin-Gal4 binding domain and the Gal4 activation domain, or the Gal4 binding domain and the BIG3-Gal4 activation domain. The p53-Gal4 binding domain and SV40LT-Gal4 activation domain were used as positive controls. (B) The table shows the activity of the nonselectable β-gal marker in yeast cells transfected with angiostatin-Gal4 binding and the BIG3-Gal4 activation domain and controls. (C) Binding of the 421 amino acid BIG3 sequence derived from yeast two-hybrid screening was verified by coprecipitation of GST-BIG3 with angiostatin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199208&req=5

Figure 1: Yeast two-hybrid screening for angiostatin-binding proteins in a human placenta cDNA library. (A) Yeast transfected with the angiostatin-Gal4 binding domain and BIG3-Gal4 activation domain grow under growth-restricted conditions (−Leu, −His, −Trp) and in the presence of 50 mM 3-amino-1, 2,4-triazol. No growth was detected in yeast transfected with both the angiostatin-Gal4 binding domain and the Gal4 activation domain, or the Gal4 binding domain and the BIG3-Gal4 activation domain. The p53-Gal4 binding domain and SV40LT-Gal4 activation domain were used as positive controls. (B) The table shows the activity of the nonselectable β-gal marker in yeast cells transfected with angiostatin-Gal4 binding and the BIG3-Gal4 activation domain and controls. (C) Binding of the 421 amino acid BIG3 sequence derived from yeast two-hybrid screening was verified by coprecipitation of GST-BIG3 with angiostatin.
Mentions: We generated a Gal4-binding domain fusion protein containing the kringle domains 1–4 of angiostatin. This fusion protein was expressed in yeast and did not activate the His or β-gal reporter genes when expressed alone or in combination with control constructs (Fig. 1A and Fig. B). Approximately 2 × 106 clones from a human term placenta cDNA library were screened with the angiostatin-Gal4 construct. Screening under selective conditions (His−, Leu−, and Trp−) generated 37 positive clones in the CG1945 yeast strain. 7 of the 37 clones displayed high β-gal activity after incubation with ONPG at 30°C for 2 h. The DNA from the colonies that contained high β-gal activity was purified and retransfected into yeast strain Y190. Three of the seven of the colonies retained activity in the new yeast strain. Sequence analysis revealed that these three clones were derived from the same gene, and the yeast-derived sequence was subsequently named BIG3. Binding of the BIG3 peptide to angiostatin was verified by coimmunoprecipitation of angiostatin with recombinant GST-tagged BIG3 domain of angiomotin (Fig. 1 C). Angiostatin binding to BIG3 was visualized with antibodies against kringle domains 1–4 by Western blot.

Bottom Line: Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells.Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility.These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics Research and Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 76 Stockholm, Sweden.

ABSTRACT
Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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Related in: MedlinePlus