Mapping of the alpha-actinin binding site within the beta 1 integrin cytoplasmic domain.
The actin cross-linking protein alpha-actinin binds to the cytoplasmic domain of the beta 1 subunit of integrin, suggesting that alpha-actinin may form a direct link between the actin cytoskeleton and the transmembrane fibronectin receptor. In this study, we have used short synthetic peptides to localize the binding site for alpha-actinin within the cytoplasmic domain of beta 1 integrin. Four 13-residue peptides were tested in both an affinity chromatographic assay and a solid-phase binding assay. The results indicated that two regions of sequence contribute to the binding of alpha-actinin: one near where the beta 1 cytoplasmic tail emerges from the membrane and a second segment located near the C terminus of the cytoplasmic tail. This binding pattern was investigated in more detail using an adaptation of the mimotope assay, in which each of the 32 overlapping sequential decapeptide segments from the beta 1 cytoplasmic domain was assembled on the head of a different plastic pin. The peptide-pin constructs were used to detect the binding of 125I-alpha-actinin. As predicted from our initial results, alpha-actinin was found to bind to two distinct clusters of peptide segments. This represents a novel use of the mimotope pin assay to map interactive sites on structural proteins.
Otey
C A
CA
Department of Cell Biology, University of North Carolina, Chapel Hill 27599.
Vasquez
G B
GB
Burridge
K
K
Erickson
B W
BW
D000185:Actinin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D003593:Cytoplasm / Q000378:metabolism; D019012:Integrin beta1; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data
D019012:Integrin beta1; D016023:Integrins; D000185:Actinin