In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.

A PCR approach was used to clone thyrotropin-releasing hormone receptors (TRH-R) from the brain and anterior pituitary of the teleost Catostomus commersoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R1 and ccTRH-R2, were identified. ccTRH-R1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the amino acid level) with the recently discovered rat TRH-R2. It is postulated that ccTRH-R2 and rat TRH-R2 are members of the same TRH-R subfamily 2. Functional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded to the application of TRH by an induction of membrane chloride currents, indicating that ccTRH-R of both subtypes are coupled to the inositol phosphate/calcium pathway. The analysis of genomic clones revealed, for the first time, both similarities and differences in the structure of TRH-R subtype genes. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is highly conserved, as it was found at an identical position in the human TRH-R1 gene. The ccTRH-R2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-R1 genes identified so far. The analysis of the gene structure of ccTRH-R and the amino acid sequence comparisons of mammalian and teleost TRH-R of both subtypes suggest that TRH receptors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evolution, possibly in invertebrates, might be the origin of ccTRH-R genes.

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances.

Ropp 120 (restrictedly overexpressed proliferation-associated protein) is a cytoplasmic protein of 120 kDa that is significantly overexpressed in mitotic cells. Protein sequencing of the immunoaffinity purified 120-kDa protein showed it to be an as yet unknown protein. DNA sequencing revealed a cDNA sequence of 3419 bases, which includes the complete coding region of ropp120 of 2943 bases (981 amino acids). Analysis of the deduced amino acid sequence showed that ropp 120 contains four WD repeats and a well-conserved consensus sequence of serine proteases. The gene encoding ropp120 (HGMW-approved gene symbol C2orf2) was assigned to chromosome 2p21-p22 by means of radiation hybrid and fluorescence in situ hybridization mapping.

Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the a subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.

Isovaleryl-CoA dehydrogenase (IVD, EC 1.3.99.10) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immune cross-reactivity, In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD, The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human TVD, and it crossreacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD, The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes.

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca2+ dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the Gi- or Gq-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.

Although few proteins have been studies as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C11 and C502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C11-->F and C502-->T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacement reduced it to (25 +/- 2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C11 and C502 in the native CSA that joins the chain ends to form a dense conformation.

The sponge Axinella polypoides contains several d-galactose binding lectins. One of the main components, lectin I was sequenced earlier, the complete sequence of the other major constituent of saline extracts, lectin II has been determined by amino acid sequencing and mass spectrometry. Both lectins have a homology of 65% to each other and both possess a disulfide loop between positions 4 and 46. As long as this loop is closed in both lectins, they can be boiled in the presence of SDS or treated with 6 mol guanidine hydrochloride without losing their hemagglutinating activity. Incubation with β-mercaptoethanol alone does not effect the carbohydrate binding capacity either. However, reduction of the disulfide bond under chaotropic conditions destroys the activity irreversibly. This disulfide loop is also an immunologically dominant epitope in both lectins, as was revealed with monospecific polyclonal antisera. Thus, sponge lectins seem to be of different origins, since three completely different structures were described: the structure of Geodia cydonium, related to the mammalian S-type lectins with one SH-group, the Axinella lectins with one disulfide loop and the Aaptos lectins I and II with 11 cysteine residues/subunit.

Head activator (HA) is a neuropeptide conserved from hydra to humans. It acts in the development of neuronal cells and is, in hydra, an important factor in head regeneration. Here we report the solubilization and purification of one head activator receptor (Kd approximately 1 nM) from a multiheaded mutant of Chlorohydra viridissima using HA affinity chromatography. Functional solubilization of the HA receptor from hydra membranes was best performed with Triton X-100 or Chaps. The addition of salt or urea and the protein concentration were important parameters in determining the yield of solubilized receptor. For affinity chromatography HA was coupled to Sepharose. The length of the spacer was optimized with respect to binding of the solubilized HA receptor. After rigorous washing a 200-kDa protein was eluted from HA Sepharose but not from control Sepharoses coupled to bradykinin or without peptide. Ligand binding was preserved in the eluate from the HA Sepharose, and a 200-kDa protein could be photoaffinity labeled. The 200-kDa protein was shown to be glycosylated mainly of the N-linked type. By Edman degradation of the purified protein sequence information was obtained for the N-terminus and after protease digestion for several internal peptides.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the alpha-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase II beta was ruled out by testing the antibodies on crude extracts from yeast cells expressing the beta-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1 ). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2 , p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67–positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.