AMPA receptor biogenesis and trafficking

AMPA-type glutamate receptors mediate the majority of fast excitatory transmission in the central nervous system. The trafficking of AMPA receptors to and from synapses alters synaptic strength and has been recognized as a central mechanism underlying various forms of synaptic plasticity. Both secretory and endocytic trafficking events seem to be driven by the subunit composition of AMPA receptor tetramers. Moreover, recent work suggests that synapses employ different tetramer combinations in response to altered synaptic input, suggesting the existence of signalling pathways that mediate remodelling of AMPA receptors. These latest developments and recent progress in elucidating the mechanisms that underlie channel assembly and trafficking are the subject of this review.     

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.     

Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization

Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein–protein interaction domains. For example, CaMKIIα coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.     

Ribosome biogenesis: of knobs and RNA processing

The synthesis of ribosomes in eukaryotes involves processing of pre-ribosomal RNA (pre-rRNA) and sequential assembly of a large number of ribosomal proteins on the rRNAs. Although we have gained tremendous insights into the processing of pre-rRNA intermediates in the last three decades, little was known about the dynamic nature of ribosome biogenesis. Only recently the development of efficient affinity-purification procedures and mass-spectrometry techniques has allowed the isolation of large pre-ribosomal complexes, which led to the identification of several ribosome assembly intermediates and a large number of novel ribosome assembly factors. In this mini-review, we summarize some of the discoveries that have been made in the field of ribosome biogenesis in the past 30 years and highlight some key aspects about what remains to be learned.     

Ribosomal RNA processing and ribosome biogenesis in eukaryotes

In eukaryotes nearly 500 rRNAs, ribosomal proteins, snoRNAs and trans-acting factors contribute to ribosome biogenesis. After more than 30 years of intense research, the incredible complexities of nucleolar function are revealed but details often remain unclear. Here we review this progress and the many intriguing questions which remain.     

Developmentally regulated alternative RNA splicing of rat brain sodium channel mRNAs.

Two rat brain Na channel alpha-subunit cDNAs, named RII and RIIA, have almost identical coding regions, with a divergence of only 36 nucleotides (0.6%) over a total length of 6015 residues. A cluster of 20 divergent residues occurs within a 90 nucleotide segment of cDNA sequence. We now demonstrate that this 90 nucleotide segment is encoded twice in the RII/RIIA genomic sequence. Furthermore, the mutually exclusive selection of these two exons is developmentally regulated. RII mRNAs are relatively abundant at birth but are gradually replaced by RIIA mRNAs as development proceeds. The two mRNAs also appear to have different regional distributions in the developing rat brain. Strikingly, although 30 amino acids are encoded by each alternative exon, only amino acid position 209 is altered between the two, specifying asparagine in RII and aspartate in RIIA. Alternative RNA splicing may modulate the RII/RIIA sodium channel properties during neuronal development.     

Developmentally regulated glycosylation of the CD8alphabeta coreceptor stalk modulates ligand binding.

The functional consequences of glycan structural changes associated with cellular differentiation are ill defined. Herein, we investigate the role of glycan adducts to the O-glycosylated polypeptide stalk tethering the CD8alphabeta coreceptor to the thymocyte surface. We show that immature CD4(+)CD8(+) double-positive thymocytes bind MHCI tetramers more avidly than mature CD8 single-positive thymocytes, and that this differential binding is governed by developmentally programmed O-glycan modification controlled by the ST3Gal-I sialyltransferase. ST3Gal-I induction and attendant core 1 sialic acid addition to CD8beta on mature thymocytes decreases CD8alphabeta-MHCI avidity by altering CD8alphabeta domain-domain association and/or orientation. Hence, glycans on the CD8beta stalk appear to modulate the ability of the distal binding surface of the dimeric CD8 globular head domains to clamp MHCI.     

Developmentally regulated proteolytic processing of a yolk glycoprotein complex in embryos of the sea urchin,Strongylocentrotus purpuratus

We have isolated a yolk glycoprotein complex from eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of these complexes and peptide mapping of their individual glycoprotein components indicate that developmental stage-specific changes in molecular composition of the complex are due to proteolytic processing events. Our data revealed that a 180 kDa glycoprotein of the egg complex is separated by a single proteolytic cleavage into intermediate glycoproteins of 115 and 76 kDa early in development. By the hatched blastula stage, each of these intermediate glycoproteins has been further processed to lower molecular weight forms: the 115 kDa protein is proteolytically clipped to a 84 kDa form, perhaps through 110 and 105 kDa intermediaries, while the 76 kDa molecule is directly processed to a 65 kDa form.     

PKC anchoring to GluR4 AMPA receptor subunit modulates PKC-driven receptor phosphorylation and surface expression

Changes in the synaptic content of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors lead to synaptic efficacy modifications, involved in synaptic plasticity mechanisms believed to underlie learning and memory formation. Early in development, GluR4 is highly expressed in the hippocampus, and GluR4-containing AMPA receptors are inserted into synapses. During synapse maturation, the number of AMPA receptors at the synapse is dynamically regulated, and both addition and removal of receptors from postsynaptic sites occur through regulated mechanisms. GluR4 delivery to synapses in rat hippocampal slices was shown to require protein kinase A (PKA)-mediated phosphorylation of GluR4 at serine 842 (Ser842). Protein kinase C (PKC) can also phosphorylate Ser842, and we have shown that PKCgamma can associate with GluR4. Here we show that activation of PKC in retina neurons, or in human embryonic kidney 293 cells cotransfected with GluR4 and PKCgamma, increases GluR4 surface expression and Ser842 phosphorylation. Moreover, mutation of amino acids R821A, K825A and R826A at the GluR4 C-terminal, within the interacting region of GluR4 with PKCgamma, abolishes the interaction between PKCgamma and GluR4 and prevents the stimulatory effect of PKCgamma on GluR4 Ser842 phosphorylation and surface expression. These data argue for a role of anchored PKCgamma in Ser842 phosphorylation and targeting to the plasma membrane. The triple GluR4 mutant is, however, phosphorylated by PKA, and it is targeted to the synapse in CA1 hippocampal neurons in organotypic rat hippocampal slices. The present findings show that the interaction between PKCgamma and GluR4 is specifically required to assure PKC-driven phosphorylation and surface membrane expression of GluR4.     

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.     

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea

ATP-gated non-selective cation channels assembled from P2X₃ receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X₃ receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X₃ expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X₃ receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.     

Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials (M. eugenii; S.crassicaudata), a reptile (C. porosus), in two species of salmonoid fishes (S. salar; O. tshawytsch), and throughout a calendar year for sea bream (S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.     

RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs.