Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

GTP-specific fab fragment-based GTPase activity assay.

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling.

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active ß1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active ß1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1.

Distinct recycling of active and inactive ß1 integrins.

Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive ß1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active ß1 integrins is higher, whereas endocytosis of the inactive ß1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF6- and early endosome antigen 1-positive compartment in an Rab4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive ß1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic.

Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence.

Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.

SHARPIN is an endogenous inhibitor of ß1-integrin activation.

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate ß1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of ß1-integrins in an RNAi screen. SHARPIN inhibited ß1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased ß1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of ß1-integrins from inactive to active conformations.

Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration.

Integrin trafficking from and to the plasma membrane controls many aspects of cell behavior including cell motility, invasion, and cytokinesis. Recruitment of integrin cargo to the endocytic machinery is regulated by the small GTPase Rab21, but the detailed molecular mechanisms underlying integrin cargo recruitment are yet unknown. Here we identify an important role for p120RasGAP (RASA1) in the recycling of endocytosed α/ß1-integrin heterodimers to the plasma membrane. Silencing of p120RasGAP attenuated integrin recycling and augmented cell motility. Mechanistically, p120RasGAP interacted with the cytoplasmic domain of integrin α-subunits via its GAP domain and competed with Rab21 for binding to endocytosed integrins. This in turn facilitated exit of the integrin from Rab21- and EEA1-positive endosomes to drive recycling. Our results assign an unexpected role for p120RasGAP in the regulation of integrin traffic in cancer cells and reveal a new concept of competitive binding of Rab GTPases and GAP proteins to receptors as a regulatory mechanism in trafficking.

RPGR and RP2: targets for the treatment of X-linked retinitis pigmentosa?

Retinitis pigmentosa is the most important hereditary eye disease and there is currently no cure available. Although mutations were found in more than 40 genes in patients with retinitis pigmentosa, only two genes have thus far been found to be responsible for one of the most severe forms of the disease, X-linked retinitis pigmentosa. In this review, we highlight the current knowledge about the two gene products RPGR and RP2 and try to link genetic data from patients with functional data on the corresponding proteins. Based on the fact that recent gene therapeutic approaches for eye diseases are at a very promising stage, we discuss the potential of RPGR and RP2 as drug targets to treat retinitis pigmentosa.

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex.

Arl2 and Arl3, members of the Arf subfamily of small G proteins, are believed to be involved in ciliary and microtubule-dependent processes. Recently, we could identify RP2, responsible for a variant of X-linked retinitis pigmentosa, as the Arl3-specific GAP. Here, we have characterized Arl2/3 interactions. We show the formation of a ternary complex between Arl3, its cognate GAP RP2 and its retinal effector HRG4. This complex seems to be important for photoreceptor function.

The retinitis pigmentosa 2 gene product is a GTPase-activating protein for Arf-like 3.

The retinitis pigmentosa 2 (RP2) gene is responsible for a particular variant of X chromosome-linked eye disease. Previously, RP2 was shown to bind the GTP form of the small G protein Arf-like 3 (Arl3), thus qualifying as an effector. Here we present the Arl3-GppNHp-RP2 complex structure, which shows features resembling complexes with GTPase-activating proteins (GAPs). Biochemical analysis showing a 90,000-fold stimulation of the GTPase reaction together with the structure of an Arl3-GDP-AlF4--RP2 transition state complex showed that RP2 is an efficient GAP for Arl3, with structural features similar to other GAPs. Furthermore, the effect of mutations in patients with retinitis pigmentosa correlated with their effect on catalysis, in particular the mutation of the arginine finger of RP2. The cognate G protein-GAP pair is conserved in yeast as Cin4-Cin2, and the ability of RP2 to act as a GAP can be correlated with its ability to complement a CIN2-deletion phenotype.

Postsynaptic recruitment of Dendrin depends on both dendritic mRNA transport and synaptic anchoring.

Synaptic plasticity and memory formation involve remodeling of the postsynaptic cytoskeleton, a process that is in part based on both local translation of dendritic mRNAs and synaptic recruitment of newly synthesized proteins. The postsynaptic component Dendrin that is encoded by a dendritically localized mRNA is thought to modulate the structure of the synaptic cytoskeleton. However, molecular mechanisms that control extrasomatic Dendrin mRNA transport and postsynaptic protein recruitment are unknown. The data presented here reveal that Dendrin interacts with the cytoskeletal components alpha-actinin and Maguk with inverted orientation (MAGI) or synaptic scaffolding molecule (S-SCAM). The latter retains Dendrin in the cytoplasm of mammalian cells and prevents its nuclear import. Furthermore in neurons, postsynaptic clustering of Dendrin requires dendritic targeting of its messenger RNA (mRNA), a process that is mediated by a sequence motif within the 3' untranslated region. In summary our finding suggest that postsynaptic recruitment of Dendrin appears to critically depend on both local protein synthesis and association with the synaptic scaffolding protein MAGI/S-SCAM. Its nuclear localization capacity further points to a function in retrograde signaling from the synapse to the nucleus.

Crystal structure of the human retinitis pigmentosa 2 protein and its interaction with Arl3.

The crystal structure of human retinitis pigmentosa 2 protein (RP2) was solved to 2.1 angstroms resolution. It consists of an N-terminal beta helix and a C-terminal ferredoxin-like alpha/beta domain. RP2 is functionally and structurally related to the tubulin-specific chaperone cofactor C. Seven of nine known RP2 missense mutations identified in patients are located in the beta helix domain, and most of them cluster to the hydrophobic core and are likely to destabilize the protein. Two residues, Glu138 and the catalytically important Arg118, are solvent-exposed and form a salt bridge, indicating that Glu138 might be critical for positioning Arg118 for catalysis. RP2 is a specific effector protein of Arl3. The N-terminal 34 residues and beta helix domain of RP2 are required for this interaction. The abilitities of RP2 to bind Arl3 and cause retinitis pigmentosa seem to be correlated, since both the R118H and E138G mutants show a drastically reduced affinity to Arl3.

Characterization of KIBRA, a novel WW domain-containing protein.

In a yeast two hybrid screen with the human isoform of Dendrin (KIAA0749), a putative modulator of the postsynaptic cytoskeleton, we isolated a cDNA coding for a novel protein, KIBRA, possessing two amino-terminal WW domains, an internal C2-like domain and a carboxy-terminal glutamic acid-rich stretch. Northern blot analysis revealed that the expression of KIBRA mRNA was predominately found in kidney and brain. In vitro interaction studies revealed that the first KIBRA WW domain binds specifically to PPxY motifs. Transient transfection of monkey kidney cells with constructs encoding Myc-tagged KIBRA displayed a cytoplasmic localization and a perinuclear enrichment of the protein.