The recruitment of clathrin to the membrane and its assembly into coated pits results from its interaction with endocytic adaptors and other regulatory proteins in the context of a specific lipid microenvironment. Dab2 (disabled 2) is a mitotic phosphoprotein and a monomeric adaptor for clathrin-mediated endocytosis. In the present study, we employed GFP (green fluorescent protein) fusion constructs of different isoforms and mutants of rat Dab2 and characterized their effect on the size, distribution and dynamics of clathrin assemblies. Enhanced levels of expression of the p82 isoform of Dab2 in COS7 cells induced enlarged clathrin assemblies at the plasma membrane. p82–clathrin assemblies, which concentrate additional endocytic proteins, such as AP2 (adaptor protein 2) and epsin, are dynamic structures in which both p82 and clathrin exchange actively between the membrane-bound and cytosolic sub-populations. The ability of p82 to induce enlarged clathrin assemblies is dependent on the presence of a functional PTB domain (phosphotyrosine-binding domain), on binding to clathrin and phospholipids, and on a newly identified and evolutionarily conserved poly-lysine stretch which precedes the PTB domain. These same molecular features are required for Dab2 to enhance the spreading of COS7 cells on fibronectin. The ability of the p82 isoform of Dab2 to enhance cell spreading was confirmed in both HeLa cells and HBL cells (human breast epithelial cells). COS7 cells expressing GFP–p82 and plated on to fibronectin concentrate the β1 integrin into clathrin–p82 assemblies. Furthermore, during cell spreading, p82–clathrin assemblies concentrate at the site of the initial cell–matrix contact and are absent from regions of intense membrane ruffling. We propose a role for Dab2 and clathrin in integrin-mediated cell spreading.
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Research Article|
February 25 2009
Dab2 regulates clathrin assembly and cell spreading
David Chetrit;
David Chetrit
1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
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Naomi Ziv;
Naomi Ziv
1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
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Marcelo Ehrlich
Marcelo Ehrlich
1
1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
1To whom correspondence should be addressed (email marceloe@tauex.tau.ac.il).
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Publisher: Portland Press Ltd
Received:
June 25 2008
Revision Received:
November 05 2008
Accepted:
November 11 2008
Accepted Manuscript online:
November 11 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (3): 701–715.
Article history
Received:
June 25 2008
Revision Received:
November 05 2008
Accepted:
November 11 2008
Accepted Manuscript online:
November 11 2008
Citation
David Chetrit, Naomi Ziv, Marcelo Ehrlich; Dab2 regulates clathrin assembly and cell spreading. Biochem J 15 March 2009; 418 (3): 701–715. doi: https://doi.org/10.1042/BJ20081288
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