Ion channel targeting in neurons

Electrical signaling by neurons depends on the precisely ordered distribution of a wide variety of ion channels on the neuronal surface. The mechanisms underlying the targeting of particular classes of ion channels to specific subcellular sites are poorly understood. Recent studies have identified a new class of protein-protein interaction mediated by PDZ domains, protein binding modules that recognize specific sequences at the C terminus of membrane proteins. The PDZ domains of a family of synaptic cytoskeleton-associated proteins, typified by PSD-95, bind to the intracellular C-terminal tails of NMDA receptors and Shaker-type K+ channels. This interaction appears to be important in the clustering and localization of these ion channels at synaptic sites. Recognition of specific C-terminal peptide sequences by different PDZ domain-containing proteins may be a general mechanism for differential targeting of proteins to a variety of subcellular locations.     

Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae

Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.     

Osteogenic protein-1 up-regulation of the collagen X promoter activity is mediated by a MEF-2-like sequence and requires an adjacent AP-1 sequence

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (-310/-278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-beta2 or activin, which up-regulates the expression of a transforming growth factor-beta-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (-294/-285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (-310/-298, TTAAAAATAAAAA) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2-like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.     

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.     

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.     

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time.     

Interactions of the C terminus of metabotropic glutamate receptor type 1alpha with rat brain proteins: evidence for a direct interaction with tubulin

Metabotropic glutamate receptors (mGluRs) are coupled to G protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1alpha, rat brain proteins which interact with the intracellular carboxy terminus of mGluR1alpha have been characterized, using affinity chromatography on a glutathione S-transferase fusion protein that contains the last 86 amino acids of mGluR1alpha. Three of the proteins specifically eluted from the affinity column yielded protein sequences, two of which were identified as glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin; the other was an unknown protein. The identity of tubulin was confirmed by western immunoblotting. Using a solid-phase binding assay, the mGluR1alpha-tubulin interaction was shown to be direct, specific, and saturable with a KD of 2.3+/-0.4 microM. In addition, mGluR1alpha, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti-beta-tubulin antibodies. However, mGluR1alpha could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl-terminal tail of mGluR1alpha are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.     

Species-specific binding of sperm proteins to the extracellular matrix (zona pellucida) of the egg

The zona pellucida is an extracellular matrix surrounding the mammalian egg where species-specific gamete recognition and signaling occur. Pig zona pellucida were isolated in large amounts and used as an affinity matrix for detergent-solubilized, biotinylated membrane proteins of pig spermatozoa. On non-reducing SDS-polyacrylamide gel electrophoresis, specifically bound sperm proteins migrated with M(r) 170,000, 150,000, 130,000, 56,000, and 50,000 (p50). Disulfide bond reduction separated each of the M(r) 130,000-170,000 proteins into M(r) 105,000 (p105) and M(r) 45,000 (p45) subunits, indicating that these high M(r) proteins are related. Based on two-dimensional electrophoresis, the M(r) 56,000 band was composed of three to four proteins that migrated with M(r) 56,000-62,000 (p56-62) in the second (reducing) dimension. p50 bound to heterologous zona pellucida (murine, bovine) and to Xenopus laevis oocyte envelopes, demonstrating a lack of species specificity to its binding and was identified as proacrosin/acrosin based on amino acid sequences of two tryptic peptides and its interaction with monospecific antibodies to proacrosin. In contrast, p105/p45 and one or more of the p56-62 proteins bound to pig zona pellucida but not to the egg extracellular matrices of the other species; these proteins therefore exhibited the species-specific binding to the zona pellucida expected for molecules involved in specific gamete adhesion. Amino acid sequences of nine tryptic peptides derived from p105/p45 did not match peptide sequences in existing databases, establishing it as a unique protein. These (p105/p45 and at least one p56-62 protein) are the first sperm membrane proteins to be identified that bind in a species-specific manner to the egg extracellular matrix.     

Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains

Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.     

Involvement of selectin-like carbohydrate binding specificity in human gamete interaction

The recognition of carbohydrate epitopes by complimentary protein receptors has been shown to be a critical factor in gamete interaction in many different animal species. In this study it was hypothesized that, in the human, gamete binding requires an interaction between selectin ligands on the zona pellucida and putative egg binding proteins on the sperm surface. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of sperm to the zona) and advanced methods of carbohydrate analysis were used to test this hypothesis. From these tests it was shown that oligosaccharide recognition is also required for initial human gamete binding. This study suggests the existence of distinct egg binding proteins on human sperm that can bind to selectin ligands. Additionally, the results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Glycoconjugates that manifest selectin-ligand activity and that express specific carbohydrate epitopes have potent contraceptive and immunosuppressive effects. Such specific oligosaccharide sequences may provide an appropriate recognition signal for embryo development and protection.     

Identification of a novel mRNA-associated protein in oocytes of Pleurodeles waltl and Xenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the complete Pleurodeles cDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS-PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)+ RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein-protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinant Pleurodeles RAP55 fragment recognize the protein from Pleurodeles and Xenopus. The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.