Characterization of an actin-binding site within the talin FERM domain.
Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.
Lee
Ho-Sup
HS
Muscle Biology Group, Departments of Biochemistry, Biophysics, and Molecular Biology and of Animal Science, Iowa State University, Ames, IA 50011-3260, USA.
Bellin
Robert M
RM
Walker
Diane L
DL
Patel
Bipin
B
Powers
Pam
P
Liu
Hongjun
H
Garcia-Alvarez
Begoña
B
de Pereda
José M
JM
Liddington
Robert C
RC
Volkmann
Niels
N
Hanein
Dorit
D
Critchley
David R
DR
Robson
Richard M
RM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002645:Chickens; D002522:Chlorocebus aethiops; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016608:Talin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016608:Talin; D017853:Phosphotransferases (Alcohol Group Acceptor)