Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

被引:53
作者
Mateer, SC
Morris, LE
Cromer, DA
Benseñor, LB
Bloom, GS
机构
[1] Univ Virginia, Dept Biol, Charlottesville, VA 22903 USA
[2] Univ Virginia, Dept Cell Biol, Charlottesville, VA 22903 USA
来源
CELL MOTILITY AND THE CYTOSKELETON | 2004年 / 58卷 / 04期
关键词
cell motility; lammelipodium; Cdc42; Rac1; calmodulin;
D O I
10.1002/cm.20013
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 muM K-d) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K-d of similar to47 muM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. (C) 2004 Wiley-Liss, Inc.
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收藏
页码:231 / 241
页数:11
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