Integrin ␣8␤1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of ␣8␤1 integrin toward a panel of RGD-containing ligands. Integrin ␣8␤1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ؎ 0.01 nM, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to ␣8␤1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to ␣8␤1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrinligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of ␣8␤1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIF-EIER sequence was strongly inhibitory, and was ϳ2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to ␣8␤1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to ␣8␤1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).2 They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed ␣ and ␤. In mammals, 18 ␣ and 8 ␤ subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers.Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen-and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include ␣5␤1, ␣8␤1, ␣IIb␤3, and ␣V-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities. The ␣8 integrin subunit was originally identified in chick nerves (4). Integrin ␣8␤1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis.

␣v␤8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-␤1. We comprehensively determined the binding activity of the ␣v␤8 integrin toward 25 secreted proteins having an RGD motif. The ␣v␤8 integrin strongly bound to latent TGF-␤1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-␤1 demonstrated that the high affinity binding of ␣v␤8 integrin to latent TGF-␤1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-␤1. Consistent with the critical role of Leu-218 in latent TGF-␤1 recognition by ␣v␤8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-␤1 with ␣v␤8 integrin, whereas a 9-mer peptide with an RGDA sequence was ϳ60-fold less inhibitory. Because ␣v␤3 integrin did not exhibit strong binding to latent TGF-␤1 or distinguish between RGDL-and RGDA-containing peptides, we explored the mechanism by which the integrin ␤8 subunit defines the high affinity binding of latent TGF-␤1 by ␣v␤8 integrin. Production of a series of swap mutants of integrin ␤8 and ␤3 subunits indicated that the high affinity binding of ␣v␤8 integrin with latent TGF-␤1 was ensured by interactions between the Leu-218 residue and the ␤8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of ␣v␤8 integrin toward latent TGF-␤1. In support of this conclusion, high affinity binding toward the ␣v␤8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence.Integrins are a family of adhesion receptors that bind to a variety of extracellular ligands, typically cell adhesion proteins in the extracellular matrix (ECM).2 Integrins play mandatory roles in embryonic development and the maintenance of tissue architecture by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed ␣ and ␤. In mammals, 18 ␣ and 8 ␤ subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers, among which 18 isoforms function as ECM receptors. Based on their ligand binding specificities, ECM-binding integrins are classified into three major groups as follows: laminin-, collagen-, and Arg-Gly-Asp (RGD)-binding integrins (1, 2), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include ␣5␤1, ␣8␤1, ␣IIb␤3, and ␣v-containing integrins, which interact with a variety of ECM ligands containing RGD motifs with distinct binding specificities.The integrin ␣v subunit was originally identified as a receptor for vitronectin (3). The ␣v-containing integrins are widely expressed on many cell types, including neural crest cells, glial cells, muscle cells, osteoclasts, epithelial cells, and vascular endothelial cells during embryonic development (4...

Western high‐fat diets (HFD) are regarded as a major risk factor for prostate cancer (PCa). Using prostate‐specific Pten‐knockout mice as a PCa model, we previously reported that HFD promoted inflammatory PCa growth. The composition of the gut microbiota changes under the influence of diet exert various effects on the host through immunological mechanisms. Herein, we investigated the etiology of HFD‐induced inflammatory cancer growth and the involvement of the gut microbiome. The expression of Hdc, the gene responsible for histamine biosynthesis, and histamine levels were upregulated in large prostate tumors of HFD‐fed mice, and the number of mast cells increased around the tumor foci. Administration of fexofenadine, a histamine H1 receptor antagonist, suppressed tumor growth in HFD‐fed mice by reducing the number of myeloid‐derived suppressor cells and suppressing IL6/STAT3 signaling. HFD intake induced gut dysbiosis, resulting in the elevation of serum lipopolysaccharide (LPS) levels. Intraperitoneal injection of LPS increased Hdc expression in PCa. Inhibition of LPS/Toll‐like receptor 4 signaling suppressed HFD‐induced tumor growth. The number of mast cells increased around the cancer foci in total prostatectomy specimens of severely obese patients. In conclusion, HFD promotes PCa growth through histamine signaling via mast cells. Dietary high‐fat induced gut dysbiosis might be involved in the inflammatory cancer growth.