Molecular Adaptor Research Articles

The fragile X mental retardation protein is a molecular adaptor between the neurospecific KIF3C kinesin and dendritic RNA granules

Fragile X mental retardation 1 protein (FMRP) is an RNA-binding protein whose absence results in the fragile X syndrome, the most common inherited form of mental retardation. FMRP contains multiple domains with apparently differential affinity to mRNA and interacts also with protein partners present in ribonucleoprotein complexes called RNA granules. In neurons, these particles travel along dendrites and axons to translocate mRNAs to specific destinations in spines and growth cones, where local synthesis of neuro-specific proteins is taking place. However, the molecular mechanisms of how RNA granules are translocated to dendrites remained unknown. We report here the identification and characterization of the motor protein KIF3C as a novel FMRP-interacting protein. In addition, using time-lapse videomicroscopy, we studied the dynamics and kinetics of FMRP-containing RNA granules in dendrites and show that a KIF3C dominant-negative impedes their distal transport. We therefore propose that, in addition to modulate the translation of its mRNA targets, FMRP acts also as a molecular adaptor between RNA granules and the neurospecific kinesin KIF3C that powers their transport along neuronal microtubules.

Structure and evolution of RIM-BP genes: Identification of a novel family member

RIM-binding proteins (RIM-BPs) were identified as binding partners of the presynaptic active zone proteins RIMs as well as for voltage-gated Ca 2+-channels. They were suggested to form a functional link between the synaptic-vesicle fusion apparatus and Ca 2+-channels. Here we show that the RIM-BP gene family diversified in different stages during evolution, but retained their unique domain structure. While invertebrate genomes contain one, and vertebrates include at least two RIM-BPs, we identified an additional gene, RIM-BP3, which is exclusively expressed in mammals. RIM-BP3 is encoded by a single exon of which three copies are present in the human genome. All RIM-BP genes encode proteins with three SH3-domains and two to three fibronectin III repeats. The flanking regions diverge in size and sequence and are alternatively spliced in RIM-BP1 and - 2. Quantitative real-time RT-PCR and in situ hybridization analyses revealed overlapping but distinct expression patterns throughout the brain for RIM-BP1 and - 2, while RIM-BP3 was detected at high levels outside the nervous system. The modular domain structure of RIM-BPs, their expression pattern and the conservative expansion during evolution shown here support their potential role as important molecular adaptors.

Is the c‐Cbl Proto‐Oncogene Involved in Chronic Lymphocytic Leukemia?

Chronic lymphocytic leukemia (CLL) is characterized by survival advantage and accumulation of CD5+ mature B lymphocytes. Expression of zeta-chain-associated protein-70 (ZAP-70), normally present in T lymphocytes or immature B cells, is associated with disease aggressiveness, as IgVH mutational status, and some proteins implicated in survival signal pathways are found to be constitutively activated in CLL cells. ZAP-70 signaling is regulated through molecular adaptors, such as the proto-oncogene product c-Casitas B lineage lymphoma (c-Cbl). The aim of this study was to determine the implication of this proto-oncogene product in CLL in survival signals. It appeared that expression of c-Cbl was increased in CLL and not correlated to that of B cell linker protein or ZAP-70. Furthermore, c-Cbl was significantly hypophosphorylated in progressive disease, so that hypophosphorylated form of c-Cbl (c-Cbl.P) along with ZAP-70, set a cutoff ratio distributing patients with stable situation below 1, and those with progressive disease equal or above 1. Given that phospholipase gamma 2 (PLC gamma 2) function is also influenced by c-Cbl hypophosphorylation, the ratio of PLC gamma 2 to c-Cbl.P was measured in CLL B cells and consistently found to be >or= 1 in Binet stage B CLL patients, as opposed to stage A CLL patients. These findings invite analysis of the role of c-Cbl in CLL.

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Glycosylphosphatidylinositol-anchored T-cadherin (T-cad) influences several parameters of angiogenesis including endothelial cell (EC) differentiation, migration, proliferation, and survival. This presupposes signal transduction networking via mediatory regulators and molecular adaptors since T-cad lacks transmembrane and cytosolic domains. Here, using pharmacological inhibition of PI3K, adenoviral-mediated T-cad-overexpression, siRNA-mediated T-cad-depletion, and agonistic antibody-mediated ligation, we demonstrate signaling by T-cad through PI3K-Akt-GSK3beta pathways in EC. T-cad-overexpressing EC exhibited increased levels and nuclear accumulation of active beta-catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3beta (S9A-GSK3beta) abrogated the stimulatory effects of T-cad on active beta-catenin accumulation, proliferation, and survival. Integrin-linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3beta, was considered a candidate signaling mediator for T-cad. T-cad was present in anti-ILK immunoprecipitates, and confocal microscopy revealed colocalization of T-cad and ILK within lamellipodia of migrating cells. ILK-siRNA abolished T-cad-dependent effects on (Ser-473)Akt/(Ser-9)GSK3beta phosphorylation, active beta-catenin accumulation, and survival. We conclude ILK is an essential mediator for T-cad signaling via Akt and GSK3beta in EC. This is the first demonstration that ILK can regulate inward signaling by GPI-anchored proteins. Furthermore, ILK-GSK3beta-dependent modulation of active beta-catenin levels by GPI-anchored T-cad represents a novel mechanism for controlling cellular beta-catenin activity.

Dynamic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Competitive Interactions of Molecular Adaptors

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.

The use of bioluminescence resonance energy transfer for the study of therapeutic targets: application to tyrosine kinase receptors

During recent years, the bioluminescence resonance energy transfer (BRET) methodology has emerged as a powerful technique for the study of protein–protein interactions. This review focuses on recent work demonstrating the power of BRET for the study of tyrosine kinase receptors, using insulin and IGF-1 receptors as models. The authors show that BRET can be used to monitor ligand-induced conformational changes within homodimeric insulin and IGF-1 receptors, as well as heterodimeric insulin/IGF-1 hybrid receptors. BRET can also be used to study, in real time and in living cells, the interaction of tyrosine kinase receptors with cellular partners negatively or positively involved in the regulation of intracellular signalling (protein tyrosine phosphatases, molecular adaptors). In addition, BRET can be used to develop high-throughput screening assays for the search of molecules with therapeutic interest and could, therefore, constitute a valuable tool for laboratories involved in drug discovery.

Fibrillin-1 Interactions with Fibulins Depend on the First Hybrid Domain and Provide an Adaptor Function to Tropoelastin

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.

Proline-, Glutamic Acid-, and Leucine-Rich Protein-1/Modulator of Nongenomic Activity of Estrogen Receptor Enhances Androgen Receptor Functions through LIM-Only Coactivator, Four-and-a-Half LIM-Only Protein 2

Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a coregulator of multiple nuclear receptors. Molecular mechanisms of PELP1 function are not completely understood, but its expression is up-regulated in hormonal-dependent cancers. Using a yeast two-hybrid screen, we found that four-and-a-half LIM-only protein 2 (FHL2) interacted with PELP1. FHL2 is a transcriptional regulator that associates with nuclear cofactors, including androgen receptors (ARs), and contains an intrinsic activation domain. PELP1 and FHL2 interact in vitro and in vivo and colocalize in the nuclear compartment. PELP1 interacts with FHL2 via LIM domains 3 and 4 and synergistically enhances the transcriptional activity of FHL2. Src kinase is required for PELP1-mediated enhancement of FHL2 functions because knockdown of Src kinase expression or function abolished PELP1-mediated FHL2 activation functions. PELP1 interacted with AR and enhanced FHL2-mediated AR transactivation functions. PELP1 knockdown by small interfering RNA or PELP1 mutant, which lacks an activation domain, reduced FHL2-mediated AR transactivation. Biochemical analyses revealed a complex consisting of PELP1, FHL2, and AR in prostate cancer cells. PELP1/MNAR expression was elevated in high-grade prostate tumors. Our results suggest that PELP1 functions as a molecular adaptor, coupling FHL2 with nuclear receptors, and PELP1-FHL2 interactions may have a role in prostate cancer progression.

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.

Structural Determinants of Human FANCF Protein That Function in the Assembly of a DNA Damage Signaling Complex

Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.

A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

CUL4–DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation

The CUL4-DDB1-ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4-DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4-DDB1 (ref. 8). We found that CUL4A-DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4-DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.

Interaction between the insulin receptor and Grb14: A dynamic study in living cells using BRET

Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression

The IkappaB-inducing kinase (IKK) is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. IKK-regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKalpha subunit, which regulates the stability of the cyclin-dependent kinase inhibitor p27(Kip1). Increased p27(Kip1) protein levels following the transfection of IKKalpha-specific siRNAs are a result of the downregulation of the F-box protein S-phase kinase-associated protein 2 (skp2). Additionally, we demonstrate that IKKalpha signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB-containing NF-kappaB complex. Together, this work defines a novel IKKalpha-regulated growth pathway involving the p52/RelB-dependent transcriptional regulation of the skp2 gene.

An electrochemically tuneable cyclodextrin-based molecular adapter

We report the synthesis of a cyclodextrin-based molecular adapter which has the propensity to form an electrochemically tuneable ternary complex with cyclophanes 2 or 3 and ferrocene 4 in non-aqueous and aqueous environments, respectively.

Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response

A decade of intensive investigation of coactivators and corepressors required for regulated actions of DNA-binding transcription factors has revealed a network of sequentially exchanged cofactor complexes that execute a series of enzymatic modifications required for regulated gene expression. These coregulator complexes possess "sensing" activities required for interpretation of multiple signaling pathways. In this review, we examine recent progress in understanding the functional consequences of "molecular sensor" and "molecular adaptor" actions of corepressor/coactivator complexes in integrating signal-dependent programs of transcriptional responses at the molecular level. This strategy imposes a temporal order for modifying programs of transcriptional regulation in response to the cellular milieu, which is used to mediate developmental/homeostatic and pathological events.

Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

YfhJ, a Molecular Adaptor in Iron-Sulfur Cluster Formation or a Frataxin-like Protein?

The yfhJ gene is part of the isc operon, which encodes the machinery devoted to assemble iron-sulfur clusters in prokaryotes. Its transcript is a small acidic protein that binds the desulfurase IscS, which is essential in iron-specific metabolic pathways. To understand its cellular role, we have characterized the structure of YfhJ in solution and its interactions with potential cellular partners. It contains a modified winged helix motif, usually present in DNA binding proteins, and is able to bind iron cations. The IscS interaction surface is the same as that involved in iron binding. This observation and the pattern of conservation through species strongly suggest that YfhJ is a molecular adaptor that is able to modulate the function of IscS in iron-sulfur cluster formation. The remarkable similarity between the in vitro behavior of YfhJ and that of the protein frataxin also suggests new hypotheses regarding the functional role of both proteins.

TLR3 Can Directly Trigger Apoptosis in Human Cancer Cells

TLRs function as molecular sensors to detect pathogen-derived products and trigger protective responses ranging from secretion of cytokines that increase the resistance of infected cells and chemokines that recruit immune cells to cell death that limits microbe spreading. Viral dsRNA participate in virus-infected cell apoptosis, but the signaling pathway involved remains unclear. In this study we show that synthetic dsRNA induces apoptosis of human breast cancer cells in a TLR3-dependent manner, which involves the molecular adaptor Toll/IL-1R domain-containing adapter inducing IFN-beta and type I IFN autocrine signaling, but occurs independently of the dsRNA-activated kinase. Moreover, detailed molecular analysis of dsRNA-induced cell death established the proapoptotic role of IL-1R-associated kinase-4 and NF-kappaB downstream of TLR3 as well as the activation of the extrinsic caspases. The direct proapoptotic activity of endogenous human TLR3 expressed by cancerous cells reveals a novel aspect of the multiple-faced TLR biology, which may open new clinical prospects for using TLR3 agonists as cytotoxic agents in selected cancers.

Toward Single Molecule DNA Sequencing: Direct Identification of Ribonucleoside and Deoxyribonucleoside 5‘-Monophosphates by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter

Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.