Dab2 regulates clathrin assembly and cell spreading.
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 beta1 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.
Chetrit
David
D
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Ziv
Naomi
N
Ehrlich
Marcelo
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology*; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000502:physiology*; D020033:Protein Isoforms / Q000502:physiology; D051381:Rats
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; D002966:Clathrin; C478773:Dab2 protein, mouse; C501404:Dab2 protein, rat; D010750:Phosphoproteins; D020033:Protein Isoforms