PubMed Health⌕ Search

Biomedical subjects

B Diefenbach

Publications and source records attributed to B Diefenbach.

7 recordsLinked to original sources
PubMed2001

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Amino Acid Motifs↗
PubMed2000

Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation.

The physiological inertness of synthetic implant materials often results in insufficient implant integration and limited acceptance of implants in tissues. After implantation the implant surface is often separated from the surrounding healthy and regenerating tissue, for example by a fibrous capsule. To avoid this host-versus-graft reaction, a strong mechanical contact between tissue and implant must be ensured. An enhanced contact between graft and the surrounding tissue can be provided by coating the implant with cell-adhesive molecules. The highly active and alpha(v)beta(3)- and alpha(v)beta(5)-integrin-selective peptide c(-RGDfK-) (f=D-phenylalanine) was functionalized with various linker molecules containing an acrylamide end group by using the lysine side chain of c(-RGDfK-). The acrylamide group can be used to bind the peptide covalently to poly(methyl methacrylate) (PMMA) surfaces. The coated surfaces effectively bind to murine osteoblasts as well as human osteoblasts in vitro when a minimum distance of 3.5 nm between surface and the constrained RGD sequence is provided. In contrast to osteoblasts in cell suspension, surface-bound osteoblasts show no apoptosis but proliferate by a factor of 10 over a 22 d period. Coating of inert implant surfaces with highly active and alpha(v)-selective peptides affords a marked improvement in osteoblast binding over current technologies. In vivo studies show that peptide-coated PMMA pellets implanted into the patella groove of rabbits are integrated into the regenerating bone tissue faster and more strongly than uncoated pellets.

Animals↗
PubMed1999

Definition of an unexpected ligand recognition motif for alphav beta6 integrin.

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 alphav beta6, 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 alphav beta6. We compared the recognition profiles of recombinant alphav beta6 and alphav beta3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to alphav beta3 contained ubiquitous RGD sequences. However, on alphav beta6, 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 alphav beta6 in isolated receptor assays (IC50 = 20 nM), and in cell adhesion assays (IC50 = 50 microM). DLXXL peptides were highly specific inhibitors of alphav beta6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- 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 alphav beta6. The DLXXL sequence is present in several matrix components and in the beta 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 alphav beta6 which has been unrecognized previously.

Amino Acid Sequence↗
PubMed1999

Radiolabeled alpha(v)beta3 integrin antagonists: a new class of tracers for tumor targeting.

UNLABELLED: The alpha(v)beta3 integrins play an important role during tumor metastasis and tumor-induced angiogenesis. Targeting of this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Cyclo(-Arg-Gly-Asp-D-Phe-Val-) has been shown to be a selective alpha(v)beta3 integrin antagonist with high affinity. In this study we describe the synthesis and biological evaluation of [125I]-3-iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) ([125I]P2), [125I]-3-iodo-Tyr5-cyclo(-Arg-Gly-Asp-D-Phe-Tyr-) ([125I]P4) and the negative control peptide [1251]-3-iodo-D-Tyr4-cyclo(-Arg-D-Ala-Asp-Tyr-Val-) ([125I]P6). METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl amino acid coupling protocols. Radioiodination was performed using the iodogen method. The in vitro binding assays were performed using isolated, immobilized alphaIIbeta3 and alpha(v)beta3 integrins. Expression of the alphaVbeta3 receptor on the different tumors was validated by immunohistochemical methods using alpha(v) and alpha(v)beta3 specific antibodies. For biodistribution studies, nude mice with melanoma M21 or mammary carcinoma MaCaF and BALB/c mice with osteosarcoma were used. RESULTS: The in vitro binding assays demonstrate that the introduction of tyrosine and subsequent iodination have no influence on the high affinity and selectivity for alpha(v)beta3. Immunohistochemical staining clearly indicates the presence of the alpha(v)beta3 integrins on the tumor tissue of the melanoma and the osteosarcoma. Pretreatment and displacement studies show specific binding of [125I]P2 on melanoma M21-bearing nude mice and osteosarcoma-bearing BALB/c mice but less specific binding on mammary carcinomas. [125I]P2 exhibits fast elimination kinetics. The accumulation in the tumor 10 min postinjection is 2.07 +/- 0.32 %ID/g for the melanoma M21 and 3.50 +/- 0.49 %ID/g for the osteosarcoma and decreases to 1.30 +/- 0.13 %ID/g and 2.03 +/- 0.49 %ID/g 60 min postinjection, respectively. [125I]P4 shows even faster elimination kinetics, resulting in a tumor accumulation of 0.40 +/- 0.10 %ID/g 60 min postinjection for the osteosarcoma-bearing BALB/c mice. Both peptides reveal predominately hepatobiliary excretion. For [1251]P2, this also is confirmed by autoradiography. The negative control peptide [125I]P6 shows no specific activity accumulation. CONCLUSION: [125I]P2 exhibits high affinity and selectivity for the alpha(v)beta3 integrin in vitro and in vivo and, thus, represents the first radiolabeled alpha(v)beta3 antagonist for the investigation of angiogenesis and metastasis in vivo.

Animals↗
PubMed1998

Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity.

Amino Acid Sequence↗
PubMed1998

Design, synthesis and biological evaluation of nonpeptide integrin antagonists.

Recent studies demonstrated that peptide and antibody antagonists of integrin alpha v beta 3 block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/alpha-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (alpha v beta 3, alpha IIb beta 3, and alpha v beta 5) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of alpha IIb beta 3 (IC50 = 14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 micrograms/embryo).

Allantois↗