Outer Dense Fiber 2 Is a Widespread Centrosome Scaffold Component Preferentially Associated with Mother Centrioles: Its Identification from Isolated Centrosomes

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ∼90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components

In haploid Saccharomyces cerevisiae, the mating and invasive growth (IG) pathways use the same mitogen-activated protein kinase kinase kinase kinase (MAPKKKK, Ste20), MAPKKK (Ste11), MAPKK (Ste7), and transcription factor (Ste12) to promote either G1 arrest and fusion or foraging in response to distinct stimuli. This exquisite specificity is the result of pathway-specific receptors, G proteins, scaffold protein, and MAPKs. It is currently not thought that the shared signaling components function under the basal conditions of vegetative growth. We tested this hypothesis by searching for mutations that cause lethality when the STE11 gene is deleted. Strikingly, we found that Ste11, together with Ste20, Ste7, Ste12, and the IG MAPK Kss1, functions in a third pathway that promotes vegetative growth and is essential in an och1 mutant that does not synthesize mannoproteins. We term this pathway the STE vegetative growth (SVG) pathway. The SVG pathway functions, in part, to promote cell wall integrity in parallel with the protein kinase C pathway. During vegetative growth, the SVG pathway is inhibited by the mating MAPK Fus3. By contrast, the SVG pathway is constitutively activated in an och1 mutant, suggesting that it senses intracellular changes arising from the loss of mannoproteins. We predict that general proliferative functions may also exist for other MAPK cascades thought only to perform specialized functions.

Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.

The solution structure of the N-terminal domain of α2-macroglobulin receptor-associated protein

The three-dimensional structure of the N-terminal domain (residues 18–112) of α2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23–34, 39–65, and 73–88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix–loop–helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering

Several scaffold proteins for neurotransmitter receptors have been identified as candidates for receptor targeting. However, the molecular mechanism underlying such receptor clustering and targeting to postsynaptic specializations remains unknown. PSD-Zip45 (also named Homer 1c/vesl-1L) consists of the NH2 terminus containing the enabled/VASP homology 1 domain and the COOH terminus containing the leucine zipper. Here, we demonstrate immunohistochemically that metabotropic glutamate receptor 1α (mGluR1α) and PSD-Zip45/Homer 1c are colocalized to synapses in the cerebellar molecular layer but not in the hippocampus. In cultured hippocampal neurons, PSD-Zip45/Homer1c and N-methyl-d-aspartate receptors are preferentially colocalized to dendritic spines. Cotransfection of mGluR1α or mGluR5 and PSD-Zip45/Homer 1c into COS-7 cells results in mGluR clustering induced by PSD-Zip45/Homer 1c. An in vitro multimerization assay shows that the extreme COOH-terminal leucine zipper is involved in self-multimerization of PSD-Zip45/Homer 1c. A clustering assay of mGluRs in COS-7 cells also reveals a critical role of this leucine-zipper motif of PSD-Zip45/Homer 1c in mGluR clustering. These results suggest that the leucine zipper of subsynaptic scaffold protein is a candidate motif involved in neurotransmitter receptor clustering at the central synapse.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Cooperative functions of the reaper and head involution defective genes in the programmed cell death of Drosophila central nervous system midline cells

In Drosophila, the chromosomal region 75C1–2 contains at least three genes, reaper (rpr), head involution defective (hid), and grim, that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, we have utilized a well defined set of embryonic central nervous system midline cells that normally exhibit a specific pattern of glial cell death. In this study we show that both rpr and hid are expressed in dying midline cells and that the normal pattern of midline cell death requires the function of multiple genes in the 75C1–2 interval. We also utilized the P[UAS]/P[Gal4] system to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone was not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induced ectopic midline cell death that resulted in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocked both normal and ectopic rpr- and hid-induced cell death. Taken together, our results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline.

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA–INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD–NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA–TRP interactions required for gating of the TRP channel in photoreceptor cells.

Characterization of an In Vitro Model of Elastic Fiber Assembly

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.

The Interaction and Colocalization of Sam68 with the Splicing-associated Factor YT521-B in Nuclear Dots Is Regulated by the Src Family Kinase p59fyn

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5–20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein–protein interaction domain. The Src family kinase p59fyn-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.