Definition of an Unexpected Ligand Recognition Motif for αvβ6 Integrin

Abstract

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including ␣v␤6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin ␣v␤6. We compared the recognition profiles of recombinant ␣v␤6 and ␣v␤3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to ␣v␤3 contained ubiquitous RGD sequences. However, on ␣v␤6, in addition to RGD-containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to ␣v␤6 in isolated receptor assays (IC 50 ‫؍‬ 20 nM), and in cell adhesion assays (IC 50 ‫؍‬ 50 M). DLXXL peptides were highly specific inhibitors of ␣v␤6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH 2 -and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with ␣v␤6. The DLXXL sequence is present in several matrix components and in the ␤ chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin ␣v␤6 which has been unrecognized previously.Integrins are a family of heterodimeric class I transmembrane receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1). They can be grouped roughly into three classes: the ␤1 series, which are ubiquitous receptors for extracellular matrix (2); the ␤2 series, which are activatable on leukocytes and are triggered during the inflammatory response (3); and the ␣v series, which bind and mediate the cell response to provisional extracellular matrices found during wound repair and other pathological processes (4).The integrins ␣5␤1 (5), ␣IIb␤3 (6), ␣8␤1 (7), ␣v␤1 (8), ␣v␤3(9), and ␣v␤6 (10) all bind the Arg-Gly-Asp-(RGD) peptide sequence in fibronectin, where it is presented in a constrained loop (11). Soluble RGD-containing peptides can inhibit the interaction of each of these integrins with fibronectin. However, to analyze the function of individual fibronectin receptors in a particular cellular environment it is useful to have more specific inhibitors, and a variety of inhibitory antibodies, modified peptides, and non-peptidic substances has been developed. However, as yet no inhibitor has been discovered specific for ␣v␤6. ␣v␤6 is a rare integrin, induced during repair processes in epithelia (10, 12). Its only known specificities are for fibronectin (10), where it can be the dominant receptor me...

High-throughput in vivo screening of targeted molecular imaging agents

“…The OBOC technique was used to prepare several peptide libraries based on the peptide sequence X 1 X 2 DLX 5 X 6 LX 8 (DLXXL is a motif that has been shown to enhance binding of peptides to the ␣ v ␤ 6 integrin) (31). The libraries were screened in vitro using a stringent double-positive cell-growthon-bead assay (32) wherein the peptidic beads were incubated with cells expressing the ␣ v ␤ 6 integrin.…”

Integrin α8β1 mediates adhesion to LAP-TGFβ1

“…For example, interaction of the disintegrin echistatin with β1 and β3 integrins involves a secondary binding determinant on the C-terminus in addition to the RGD site (Wierzbicka-Patynowski et al, 1999). Recently, the sequence DLXXL was reported as a ligand for αvβ6 (Kraft et al, 1999). A similar sequence is found adjacent to the RGD site in LAP-TGFβ1 (RGDLXXI), LAP-TGF-β3 (RGDLXXL) and adjacent to the RGD site in Foot and Mouth Disease Virus, another recently described ligand for αvβ6 (Jackson et al, 2000).…”

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor

“…The DNA for the anti-avb6 peptide was designed based on the sequence described by Kraft et al 9 of a 12-aa peptide ligand (RTDLDSLRTYTL) for avb6. The remainder of the construct has been previously described by Jensen et al 3 In brief, the final chimeric construct consists of a Kozak consensus ribosome binding sequence; granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha leader sequence; avb6-specific binding peptide Bpep; triple glycine linker; hinge Fc; CD4 transmembrane region; and the cytoplasmic domain of the CD3-z chain.…”

“…9 Goodman and co-workers have identified several peptides that bind specifically to avb6 and not other integrins. Fibronectin is the ligand for avb6, and this interaction is strongly inhibited by the peptide RTDLDSLRTYTL.…”

“…The half-maximal inhibitory concentration of the peptide-avb6 interaction is 20 nM, and is specific to this integrin, as this peptide does not affect fibronectin binding to integrins avb3, avb5 or aIIbb3. 9 Here, we describe a peptide chimeric T cell receptor based on this 12 amino acid (aa) peptide targeting moiety, and demonstrate its capacity to redirect the effector function of T cells to avb6 integrin-expressing ovarian carcinoma cells.…”