Migration of cells and degradation of the extracellular matrix (ECM) are required for efficient tumor cell invasion, but the underlying molecular mechanisms are only partially known. The PPFIA1 gene for liprin-a1 is frequently amplified in human breast cancers. We recently demonstrated that liprin-a1 is an important regulator of cell edge dynamics during motility. We show, herein, that the liprin-a1 protein is highly expressed in human breast tumors. Functional analysis shows that liprin-a1 is specifically required for the migration and invasion of highly invasive human breast cancer MDA-MB-231 cells. We used time-lapse analysis to demonstrate defects in the motility of liprin-a1-depleted cells that include a striking instability of the lamellipodia. Liprin-a1 levels altered by either RNA interference or overexpression affected also cell spreading and the number of invadopodia per cell, but not the density of invadopodia per unit of surface area. On the other hand, silencing of liprin-a1 inhibited the degradation of the ECM, whereas its overexpression enhanced degradation, resulting in significant negative or positive effects, respectively, on the area of degradation per invadopodium. Transfection of fluorescent-labeled cortactin revealed that depletion of liprin-a1 also affected the assembly and disassembly of invadopodia, with decrease of their lifetime. Our results strongly support a novel important role of liprin-a1 in the regulation of human tumor cell invasion.

Integrin activation is needed to link the extracellular matrix with the actin cytoskeleton during cell motility. Protrusion requires coordination of actin dynamics with focal-adhesion turnover. We report that the adaptor protein liprin-α1 is stably associated with the cell membrane. Lipin-α1 shows a localization that is distinct from that of activated β1 integrins at the edge of spreading cells. Depletion of liprin-α1 inhibits the spreading of COS7 cells on fibronectin by affecting lamellipodia formation, whereas its overexpression enhances spreading, and lamellipodia and focal-adhesion formation at the cell edge. Cooperation between liprin-α1 and talin is needed, because either talin or liprin depletion prevents spreading in the presence of the other protein. The effects of liprin on spreading, but not its effects in the reorganization of the cell edge, are dependent on its interaction with leukocyte common antigen-related tyrosine phosphatase receptors. Therefore, liprin is an essential regulator of cell motility that contributes to the effectiveness of cell-edge protrusion.

G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized. We found that carboxy-terminal-truncated fragments of GIT1 bind their partners with higher efficiency compared with the full-length GIT1. We have explored the hypothesis that GIT1 is regulated by an intramolecular mechanism, and we identified two distinct intramolecular interactions between the N and C terminus of GIT1. The release of these interactions increases binding of GIT1 to paxillin and liprin-alpha, and it correlates with effects on cell spreading. Analysis of cells plated on fibronectin has shown that different deletion mutants of GIT1 either enhance or inhibit spreading, depending on their subcellular localization. Moreover, although the association between betaPIX and GIT1 is insufficient to activate GIT1 binding to paxillin, binding of a PAK1 fragment including the betaPIX-binding domain enhances paxillin binding to betaPIX/GIT1, indicating that p21-activated kinase can activate the binding of paxillin to GIT1 by a kinase-independent mechanism. The release of the identified intramolecular interaction seems to be an important mechanism for the regulation of GIT1 functions.

Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvβ3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvβ3 integrin antagonists that block αvβ3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvβ3 antagonists without paradoxical effects.

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Integrins are heterodimeric, transmembrane, cell-adhesion receptors regulating cellular functions crucial for the initiation, progression, and metastasis of solid tumors. [1] Specifically, integrin avb3 plays a key role in endothelial cell survival and migration during tumor angiogenesis. [2] It has, therefore, gained attention as an attractive therapeutic target in antiangiogenic cancer therapy. [3] avb3 relays signals bi-directionally across the plasma membrane between the extracellular ligand-binding site and the cytoplasmic domains of the receptor. Signal transfer is allosterically coupled to three major equilibrium conformational states, including the 1) inactive bent state; 2) intermediate extended state with a closed headpiece, and 3) active extended form with an open headpiece [4] ( Figure S1 in the Supporting Information). Although controversy remains concerning the level of complexity in integrin conformational changes, it is generally recognized that the out-in signaling, following ligand binding, and the consequent switch from the inactive to active state is accompanied by an outwards movement of the b-hybrid domain, characterized by a swing-out angle varying between 108 and 808. [5][6][7][8] Out-in activation of avb3 by natural ligands occurs through the recognition of a tripeptidic motif arginineglycine-aspartate (RGD). This sequence is therefore a lead for developing integrin antagonists. [9] The cyclopeptide isoDGR is a new avb3-binding motif, with potential applications in the design of integrin antagonists. [10][11][12][13][14] One major problem with integrin inhibitors is their potential to activate conformational changes, which can initiate unwanted signals that induce agonist-like activities and adverse paradoxical effects. [3,15,16] In this context, drug design studies aimed at developing new integrin blockers could benefit from information defining the receptor allosteric events induced by ligand binding. Therefore, in the attempt to characterize isoDGR-based avb3 antagonists, we exploited a combination of computational and biochemical studies to describe the dynamic changes of avb3 upon ligand binding. Herein, we demonstrate that isoDGR-based cyclopeptides unexpectedly inhibit receptor allosteric activation.To characterize the interactions and the effects of RGDand isoDGR-containing cyclopeptides on avb3 conformational dynamics, we performed all-atom molecular dynamics (MD) simulations of the integrin avb3 headpiece alone and in the presence of RGDf(NMe)V, CisoDGRC, and ac CisoDGRC cyclopeptides (Figure 1; see Supporting Information for details). To increase the conformational sampling, we adopted a multicopy approach [17] performing three independent trajectories for each system for a total cumulative simulation time of 360 ns ( Figure S2a, b and S3 in the Supporting Information). The three ligands anchor to the av and b3 domains through an electrostatic clamp that exploits similar though not identical interaction patterns (Figure 1). On one hand, the Arg guanidinium groups of the three l...

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.