RGD AND OTHER RECOGNITION SEQUENCES FOR INTEGRINS
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▪ Abstract Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands. Some other integrins bind to related sequences in their ligands. The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when insolubilized onto a surface, and inhibit it when presented to cells in solution. Reagents that bind selectively to only one or a few of the RGD-directed integrins can be designed by cyclizing peptides with selected sequences around the RGD and by synthesizing RGD mimics. As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, the RGD peptides and mimics can be used to probe integrin functions in various biological systems. Drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer.Keywords:
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Integrins are a family of heterodimeric receptors, which modulate many cellular processes including growth, death, adhesion, migration, and invasion by activating several signaling pathways. Until now, 18α and 8β subunits are known in mammals to noncovalently associate and form 24 integrin heterodimers. Integrin-binding site, the RGD (arginine-glycine-aspartate) motif, is found from several important extracellular matrix proteins, which serve as adhesive integrin ligands. The RGD motif has also been found from snake venom toxins, which inhibit integrin-binding function and serve as potent integrin antagonists. Many of these proteins are potential therapeutic agents in the treatment of integrin-realted diseases because they have higher affinity than extracellular matrix proteins. Although the RGD motif is crucial for their binding to integrins, the selectivity depends on the composition of the amino acid flanking the RGD motif and C-terminal region. Recently, a region (41KKKR45T) of the disintegrin elegantin termed the “linker region” has been shown to exhibit the inhibitory activity against the synergy site of fibronectin in promoting α5β1 integrin-mediated cell adhesion. In this study we used rhodostomin (Rho), a disintegrin with a 39SRAG43K linker sequence, and trimucin (Tmu), a disintegrin with a 41KKKR45T linker sequence, as the scaffolds to study the role of different linker regions in recognizing integrins α5β1, αIIbβ3, and αvβ3. I have expressed sixteen Rho mutant proteins and one Tmu mutant protein and purified them to homogeneity. Analysis of platelet aggregation and cell adhesion assays showed that disintegrins with 41KKKR45T linker sequence exhibited 0.8-3, 3-12, and 3-5 folds increases in inhibitory activity to integrins IIb3, v3, and 51, respectively. This is consistent with our docking structure of the Rho-integrin complex that the linker region may interact with an adjacent metal-ion-dependent adhesion site of subunit through ionic interaction. Using alanine scanning mutation on the 39KKKR43T region, we found that the K40A, and T43A mutants caused 2-, 5-, and 5-, and 2.5-, 5-, and 5-folds decreases in inhibitory activity to integrins IIb3, v3, and 51, respectively. The K39A also caused 2- and 5-folds decreases in inhibitory activity to integrins v3 and 51. In contrast, the mutations on 41K and 42R have little effect on their inhibitory activity, suggesting that the 39K, 40K, and 43T residues in linker region of disintegrin may interact with integrins. We also found that the I47R mutant with 39KKKR43T exhibited 5.0-7.0-folds increases in inhibiting integrin 51, and the I47R mutant with 39SRAG43K exhibited 2.3-, 9.2-, and 24-folds-increase in inhibitory activity to integrins IIb3, v3, and 51, respectively. The analysis of the mutants containging the linker region sequences (39IEEG43T, 39KGAG43K, 39LKEG43T, and 39MKKG43T) from other disintegrins showed that the linker region sequences play an important role in their binding to integrins. Taken together, Rho mutants containing 39KKKR43T-48PRGDM53P-67Y68H, 39MKKG43T-48ARGDN53P- 67NGLY71G, and 39KAKR43A-48ARGDN53P-67NGLY71G have higher affinity for integrins V3. In addition, the mutants containing 39KKKRTICR47IARGDN53P-67NGLY71G, 39KKKRTICR47RARGD N53P-67NGLY71G, and 39SRAGKICR47RARGDN53P-67NGLY71G have higher activitiy in inhibiting integrins 51 and V3. The results of this study will serve as the basis to design potent integrins-specific disintegrins.
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The RGD motif is a cell adhesion sequence that binds to integrins, a receptor family for extracellular matrix proteins. We previously reported that the RGDX1X2 sequence, where X1X2 is VF or NY, is required for integrin αvβ5-mediated cell adhesion. However, the importance and applications of the X1X2 combinations and their surrounding sequences of integrin αvβ5-binding RGDX1X2-containing peptides have not been comprehensively elucidated. Therefore, we aimed to identify an RGD-containing peptide with enhanced integrin αvβ5 binding activity. We synthesized various peptides based on the RGDVF and RGDNY peptides to optimize the N-terminal, C-terminal, and X1X2 combinations of the RGDX1X2 sequence. These peptides were immobilized on maleimide-functionalized bovine serum albumin-coated plates via a thiol-maleimide reaction, and cell adhesion was evaluated using HeLa cells and human dermal fibroblasts. Consequently, CPPP-RGDTF and CPPP-RGDTFI were identified as highly active peptides for integrin αvβ5-mediated cell adhesion. CPPP-RGDTF and CPPP-RGDTFI are expected to serve as cell adhesion molecules for developing culture substrates and biomaterials. Furthermore, these findings provide important novel insights into the interaction between the RGD motifs and integrins.
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Abstract Integrin-mediated cell adhesion on extracellular matrix works essentially on numerous physiological processes such as cell adhesion, migration and angiogenesis. As integrin ανβ3 and integrin α5β1 are the representative receptors for cell adhesion and due to their important function in cancer biology, the antagonists targeting the integrins has been sought for long. Lately, the interactions between ADAM (A Disintegrin And Metalloprotenase) 15, the only member in ADAM family with Arg-Gly-Asp (RGD) motif, and the ανβ3 and α5β1 intergins was observed. For that, we experimented on the cellular function of recombinant ADAM15-derived disintegrin-like domain containing Asp-Typ-Lys-Arg-Gly-Asp (rNWKRGD) in integrin-mediated cell adhesion. The binding affinity of HUVEC, COS-1, MCF-7 and MDAH 2274 cells to various extracellular matrix proteins were increased by the disintegrin-like domain in a dose-dependent manner, and the presence of inhibitory integrin α5β1 antibody thoroughly abrogated the rNWKRGD-stimulated binding. By mutagenic analysis, it was founded that RGD motif is essential for the binding. In comparison, saxatilin, RGD disintegrin from snake venom, inhibited binding of cells to ECM proteins. Through flow cytometric analysis, we confirmed that β1 integrin on the cell surface was activated by rNWKGRD. All these results indicated that the activation of integrin α5β1 mediates the rNWKRGD-stimulated cell adhesion. Therefore rNWKRGD could function as the potential activator of cell migration and proliferation. Although disintegrin is known as inhibitor of cell migration and proliferation, it is possible that ADAM15 disintegrin domain could have a regulatory role in integrin function such as angiogenesis and invasion of cancer cells. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. 2009-0081759), KIST grant, and the Brain Korea 21 (BK21).) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B42.
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▪ Abstract Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands. Some other integrins bind to related sequences in their ligands. The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when insolubilized onto a surface, and inhibit it when presented to cells in solution. Reagents that bind selectively to only one or a few of the RGD-directed integrins can be designed by cyclizing peptides with selected sequences around the RGD and by synthesizing RGD mimics. As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, the RGD peptides and mimics can be used to probe integrin functions in various biological systems. Drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer.
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The integrins are a family of αβ heterodimeric membrane proteins that mediate dynamic linkages between extracellular matrix (ECM) proteins and the intracellular actin cytoskeleton.Many of integrins bind to the Arg-Gly-Asp(RGD)sequence which exists in ECM proteins and modulate many cellular processes.The RGD motif has also been found in many toxins from snake venom and other animal sources.These toxins can specifilally inhibit the binding of integrins to ECM,thus becoming the effective integrin antagonists.Here we review the integrin structure and functions,and the disintegrin structural features as antagonists of integrin function.The application of integrins and RGD motif structure as potential drug targets is also discussed.
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Integrins are the major cell surface receptors for cells to interact with extracellular matrix proteins and play an important role in cytoskeleton reorganization and signal transduction. Disintegrins are potent integrin inhibitors found in snake venoms. Depending on the size and the number of cysteines, they can be classified into small, medium, long, and dimeric disintegrins. To study structure and functional relationships of short and medium disintegrins, we expressed rhodostomin (Rho), a 68‐residue disintegrin with six disulfide bonds, and echistatin (Ech), a 49‐residue disintegrin with four disulfide bonds, in Pichia pastoris as the model proteins. The analysis of cell adhesion assay showed that Rho mutant containing ARGDDM sequences in the RGD loop and NPHKGPAT sequence in the C‐terminus exhibited 14.4‐, 4.1‐, and 2.33‐folds decrease in inhibitory activity to integrins £\IIb£]3, £\v£]3 and £\5£]1, respectively. Backbone dynamics analysis showed that their similar dynamics properties on the RGD motif. In contrast, the C‐terminal region of Rho is more flexible of that of Ech. This suggests that the dynamics properties of the C‐terminus of disintegrins also play a crucial role in determining their affinity and selectivity to integrins.
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