Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain.
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 microM 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 approximately 47 microM, 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.
Mateer
Scott C
SC
Department of Biology, University of Virginia, Charlottesville, VA, USA.
Morris
Leah E
LE
Cromer
Damond A
DA
Benseñor
Lorena B
LB
Bloom
George S
GS
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014162:Transfection; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D020703:ras GTPase-Activating Proteins; D000096985:Calponins; C101203:IQ motif containing GTPase activating protein 1