Abstract B42: Modulation of integrin-mediated cell adhesion by ADAM15 disintegrin-like domain
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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.Keywords:
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Integrins are heterodimeric trans-membrane receptor proteins that mediate the interaction of cells with extracellular matrix proteins (ECM). They mediate various growth factor dependent cell-signaling pathways during the development of fibrosis that is characteristic of all forms of chronic kidney diseases. Although integrin β1 is the most abundant integrin subunit in kidney and can form complexes with 12 different α subunits, integrin β3 is the best studied integrin β subunit and serves as the canonical model for integrin function based on the high sequence homology between the trans-membrane and cytosolic domains of integrin β subunits. A conserved lysine residue towards the C-terminus of integrin β3 subunit is reported to be important for regulating the activation of integrin αIIbβ3 complexes; however the functional importance of this lysine is unknown in β1 integrins. We investigated the role of this lysine residue in integrin β1-dependent kidney collecting duct cell function. We expressed the mutant protein where the lysine is mutated to glutamic acid in collecting duct cells null for integrin β1. Collecting duct cells expressing mutant protein had decreased the adhesion of cells to collagen IV mediated by integrin α1β1 by 80% (0.95 vs 0.18). This mutation also decreased the ability of IMCD cells adhesion to collagen I mediated by integrin α2β1 by 82% (0.78 vs 0.15). In contrast to earlier reports in integrin β3, this mutation did not significantly alter the amount of active integrin β1 on the cell surface as estimated by FACS analysis; however we did observe a decrease in conformation specific antibody binding on cells adhered to collagen (0.70 vs 0.30). We also investigated the role of this lysine residue in complex formation of purified integrin β1 with integrin α1 and α2 TM/CT domains in phospholipid bicelles using fluorescence anisotropy. The dissociation constant for binding was estimated to be >3.2 mol and mutation of lysine residue did not significantly alter their binding ability. This contrasted with integrin αIIb β3 where we found fourfold decrease in binding ability (Kd 0.09 ± 0.03 mol and 0.33 ± 0.05 mol). Our data clearly suggest that conserved transmembrane lysine in both integrin β3 and integrin β1 regulate cell functions by distinct mechanisms.
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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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ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain. In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-S y 5 y ) or astrocytoma cells (U373 and U87 MG). Analysis of ADAM 23 binding to integrins revealed a specific interaction with αvβ3, mediated by a short amino acid sequence present in its putative disintegrin loop. This sequence lacks any RGD motif, which is a common structural determinant supporting αvβ3-mediated interactions of diverse proteins, including other disintegrins. αvβ3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23. On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in αvβ3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin.
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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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Integrins are α/β heterodimeric cell surface receptor and are involved in cell proliferation, migration and cell survival. Because they contribute to the initiation and progression of many common diseases, such as thrombosis, immune system disorders, cancer, and osteoporosis, they are attractive therapeutic targets for many diseases. Disintegrins are potent integrin inhibitors found from snake venoms. According to the size and the number of disulfide bonds, they can be classified into small, medium, long, and dimeric disintegrins. To study structure, dynamics, and function relationships of short and medium disintegrins, we expressed echistatin (Ech), rhodostomin (Rho), their corresponding and C-terminal mutants in Pichia pastoris. Ech, a 49-residue short disintegrin with four disulfide bonds, and Rho, a 68-residue medium disintegrin with six disulfide bonds, are RGD-containing proteins. We successfully expressed recombinant Ech and its mutants and purified them to homogeneity with the yields of 2-7 mg/L. Recombinant Ech inhibited platelet aggregation with the IC50 value of 210.5 nM. Functional and structural analyses showed that Ech produced in P. pastoris retained its function and native fold. The replacement of Ech’s RGD loop (ARGDDM) and C-terminal (HKGPAT) sequences with Rho’s (PRGDMP) and (YH) sequences caused 7.3-, 4.5-, and 2.0-folds less active than those of Rho in inhibiting integrins αIIbβ3, α5β1, and αvβ3. The replacement of Rho’s RGD loop (PRGDMP) and C-terminal (YH) sequence with Ech’s (ARGDDM) and (HKGPAT) sequences caused 14.2-, 3.1-, and 6.7-folds less active than those of Ech in inhibiting integrins αIIbβ3, α5β1, and αvβ3. These results suggest that disintegrins scaffolds are sensitive for the activities of integrins. Backbone dynamics analysis showed that Ech and Rho with an ARGDDM motif exhibited similar dynamical properties for the RGD motif; however, C-terminus of Rho with a HKGPAT sequence was more flexible than that of Ech. Platelet aggregation analysis of Ech C-terminal mutants showed that the length of C-terminus and the K45 residue are important for interacting with integrin αIIbβ3. Ech and its C-terminal mutants can also induce ligand-induced binding site (LIBS) on integrins αIIbβ3 and αvβ3, and their activity is correlated with their inhibition activity of cell adhesion. The comparison of Ech with its C-terminal deletion mutants also showed that the length of C-terminus is important for LIBS induction. NMR analysis showed that disintegrin scaffolds affect the orientation of the D residue and dynamical properties of the RGD motif and C-terminus. Docking structure also confirmed that disintegrin scaffolds affect the orientation of C-terminus, resulting in their differences in interacting with integrins. These results indicate that disulfide patterns of disintegrins are important for the conformation of their C-terminus, resulting in their functional, structural, and dynamic differences. In addition, we found that recombinant Ech can inhibit the migration of human A375 melanoma cell. Ech can inhibit the migration of human melanoma A375 and glioblastoma U373 MG cells with the IC50 values of 1.5 and 4.9 nM. In contrast, it inhibited human pancreatic tumor cell Panc-1 with the IC50 value of 123.9 nM. The differences in its activity may be due to expression levels of integrins in cancer cells. In conclusion, our study indicates that disulfide patterns of disintegrins are important for the conformation of their RGD motif and C-terminus, resulting in their functional, structural, and dynamic differences. These findings may serve as the basis for the design of anti-tumor drugs using disintegrin scaffolds.
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The ADAM (a disintegrin-like and metalloproteinase) proteins are a family of transmembrane cell-surface proteins with important functions in adhesion and proteolytic processing in all animals. Human ADAM-15 is the only member of the ADAM family with the integrin binding motif Arg-Gly-Asp (RGD) in its disintegrin-like domain. This motif is also found in most snake venom disintegrins and other disintegrin-like proteins. This unique RGD motif within ADAM-15 serves as an integrin ligand binding site, through which it plays a pivotal role in interacting with integrin receptors, a large family of heterodimeric transmembrane glycoproteins. This manuscript will present a review of the RGD-containing disintegrin-like domain structures and the structural features responsible for their activity as antagonists of integrin function in relation to the canonical RGD template.
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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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