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 beta1 subunit of integrin, suggesting that alpha-actini n may form a direct link between the actin cytoskeleton and the transm embrane fibronectin receptor. In this study, we have used short synthe tic peptides to localize the binding site for alpha-actinin within the cytoplasmic domain of beta1 integrin. Four 13-residue peptides were t ested in both an affinity chromatographic assay and a solid-phase bind ing assay. The results indicated that two regions of sequence contribu te to the binding of alpha-actinin: one near where the beta1 cytoplasm ic tail emerges from the membrane and a second segment located near th e C terminus of the cytoplasmic tail. This binding pattern was investi gated in more detail using an adaptation of the mimotope assay, in whi ch each of the 32 overlapping sequential decapeptide segments from the beta1 cytoplasmic domain was assembled on the head of a different pla stic pin. The peptide-pin constructs were used to detect the binding o f I-125-alpha-actinin. As predicted from our initial results, alpha-ac tinin was found to bind to two distinct clusters of peptide segments. This represents a novel use of the mimotope pin assay to map interacti ve sites on structural proteins.