Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins.
Integrin cytoplasmic domains connect these receptors to the cytoskeleton. Furthermore, integrin-cytoskeletal interactions involve ligand binding (occupancy) to the integrin extracellular domain and clustering of the integrin. To construct mimics of the cytoplasmic face of an occupied and clustered integrin, we fused the cytoplasmic domains of integrin beta subunits to an N-terminal sequence containing four heptad repeat sequences. The heptad repeats form coiled coil dimers in which the cytoplasmic domains are parallel dimerized and held in an appropriate vertical stagger. In these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the heptad repeat sequences; 2) that the cytoskeletal proteins talin and filamin are among the polypeptides that bind to the integrin beta1A tail. Filamin, but not talin binding, is enhanced by the insertion of Gly spacers; 3) binding of both cytoskeletal proteins to beta1A is direct and specific, since it occurs with purified talin and filamin and is inhibited in a point mutant (beta1A(Y788A)) or in splice variants (beta1B, beta1C) known to disrupt cytoskeletal associations of beta1 integrins; 4) that the muscle-specific splice variant, beta1D, binds talin more tightly than beta1A and is therefore predicted to form more stable cytoskeletal associations; and 5) that the beta7 cytoplasmic domain binds filamin better than beta1A. The structural specificity of these associations suggests that these mimics offer a useful approach for the analysis of the interactions and structure of the integrin cytoplasmic face.
Pfaff
M
M
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Liu
S
S
Erle
D J
DJ
Ginsberg
M H
MH
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003285:Contractile Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D064448:Filamins; D005998:Glycine / Q000737:chemistry; D016023:Integrins / Q000378:metabolism*; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D016608:Talin / Q000378:metabolism*
D003285:Contractile Proteins; D064448:Filamins; D016023:Integrins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016608:Talin; D005998:Glycine