Vollum Institute

The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 A resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 A in length, that runs along the sevenfold axis and ranges from 14 A to 46 A in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel beta barrel, to which each protomer contributes two beta strands, each 65 A long. The interior of the beta barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 A wide. The structure proves the heptameric subunit stoichiometry of the alpha-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of beta barrel pore-forming toxins.

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.

CREB binding protein (CBP) and p300 were both identified initially in protein interaction assays-the former through its association with the transcription factor CREB The recognition that these two proteins, one involved in transcription and the other in cell transformation, had highly conserved sequences suggested that they had the potential to participate in a variety of cellular functions (Fig. Several excellent reviews

Melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) regulate pigmentation and adrenal cortical function, respectively. These peptides also have a variety of biological activities in other areas, including the brain, the pituitary, and the immune system. A complete understanding of the biological activities of these hormones requires the isolation and characterization of their corresponding receptors. The murine and human MSH receptors (MSH-Rs) and a human ACTH receptor (ACTH-R) were cloned. These receptors define a subfamily of receptors coupled to guanine nucleotide-binding proteins that may include the cannabinoid receptor.

Increased activity of dopamine-containing neurons in the ventral tegmental area is necessary for the reinforcing effects of opioids and other abused drugs. Intracellular recordings from these cells in slices of rat brain in vitro showed that opioids do not affect the principal (dopamine-containing) neurons but hyperpolarize secondary (GABA-containing) interneurons. Experiments with agonists and antagonists selective for opioid receptor subtypes indicated that the hyperpolarization of secondary cells involved the mu-receptor. Most principal cells showed spontaneous bicuculline-sensitive synaptic potentials when the extracellular potassium concentration was increased from 2.5 to 6.5 or 10.5 mM; these were prevented by TTX and assumed to result from action potentials arising in slightly depolarized local interneurons. The frequency of these synaptic potentials, but not their amplitudes, was reduced by opioids selective for mu-receptors. It is concluded that hyperpolarization of the interneurons by opioids reduces the spontaneous GABA-mediated synaptic input to the dopamine cells. In vivo, this would lead to excitation of the dopamine cells by disinhibition, which would be expected to contribute to the positive reinforcement seen with mu-receptor agonists such as morphine and heroin.

Since the original somatomedin hypothesis was conceived, a number of important discoveries have allowed investigators to modify the concept. Originally somatic growth was thought to be controlled by pituitary GH and mediated by circulating insulin-like growth factor-I (IGF-I, somatomedin C) expressed exclusively by the liver. With the discovery that IGF-I is produced by most, if not all, tissues, the role of autocrine/paracrine IGF-I vs. the circulating form has been hotly debated. Recent experiments using transgenic and gene-deletion technologies have attempted to answer these questions. In the liverspecific igf-1 gene-deleted mouse model, postnatal growth and development are normal despite the marked reduction in circulating IGF-I and IGF-binding protein levels; free IGF-I levels are normal. Thus, the normal postnatal growth and development in these animals may be due to normal free IGF-I levels (from as yet unidentified sources), although the role of autocrine/paracrine IGF-I has yet to be determined.

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

The peak concentration and rate of clearance of neurotransmitter from the synaptic cleft are important determinants of synaptic function, yet the neurotransmitter concentration time course is unknown at synapses in the brain. The time course of free glutamate in the cleft was estimated by kinetic analysis of the displacement of a rapidly dissociating competitive antagonist from N-methyl-D-aspartate (NMDA) receptors during synaptic transmission. Glutamate peaked at 1.1 millimolar and decayed with a time constant of 1.2 milliseconds at cultured hippocampal synapses. This time course implies that transmitter saturates postsynaptic NMDA receptors. However, glutamate dissociates much more rapidly from alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Thus, the time course of free glutamate predicts that dissociation contributes to the decay of the AMPA receptor-mediated postsynaptic current.

POMC, the precursor of ACTH, MSH, and beta-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. We report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacologically distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting a role in the central control of pituitary function. MC4-R is also unique in its expression in numerous cortical and brainstem nuclei. Together, MC3-R and/or MC-4R mRNA are found in every nucleus reported to bind MSH in the adult rat brain and define neuronal circuitry known to be involved in the control of diverse neuroendocrine and autonomic functions. The high degree of conservation, distinct pharmacology, and unique neuronal distribution of the MC4 receptor suggest specific and complex roles for the melanocortin peptides in neuroendocrine and autonomic control.

Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

The transport of ions across the membranes of cells and organelles is a prerequisite for many of life's processes. Transport often involves very precise selectivity for specific ions. Recently, atomic-resolution structures have been determined for channels or pumps that are selective for sodium, potassium, calcium, and chloride: four of the most abundant ions in biology. From these structures we can begin to understand the principles of selective ion transport in terms of the architecture and detailed chemistry of the ion conduction pathways.

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non-nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L-glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated L-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.

CREB1-binding protein (CBP) and p300 are believed to participate in the activities of hundreds of different transcription factors (see Fig.1). Current models suggest that the binding of these coactivators to transcription factor activation domains positions histone acetyltransferases (HATs) near specific nucleosomes in target gene promoter regions (for review, see Ref. 1Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1406) Google Scholar). Interactions with components of the general transcriptional machinery, such as TFIID, TFIIB, and the RNA polymerase II holoenzyme (RNAPII) have also been suggested to contribute to CBP/p300 function. The simultaneous interaction of multiple transcription factors with CBP/p300 has been proposed to contribute to transcriptional synergy. Conversely, competition for CBP/p300 binding has been suggested to mediate some examples of signal-induced transcriptional repression. An overview of CBP/p300 in cellular growth and differentiation has recently been published (2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar), but many questions regarding their role in transcriptional regulation remain unanswered. This review deals with some of the more controversial aspects of CBP/p300 function. In particular, we will ask whether CBP and p300 have distinct functions, review the evidence for their regulation by phosphorylation, and ask whether they function primarily by acetylating histones or other proteins. We will also revisit the evidence for the role of CBP/p300 as transcriptional “integrators.” Finally, we will attempt to localize CBP/p300 function within the complex series of processes involved in transcriptional activation. Although CBP and p300 are highly related and share many functional properties, there is evidence that these factors are not really interchangeable. Subtle differences in the expression of CBP and p300 during development (3Partanen A. Motoyama J. Hui C.C. Int. J. Dev. Biol. 1999; 43: 487-494PubMed Google Scholar) may explain why knockouts of the two coactivators in mice result in somewhat distinct phenotypes. For example, heterozygosity for CBP causes certain hematological defects and a predisposition to cancer that is not seen in mice lacking one allele of p300 (4Kung A.L. Rebel V.I. Bronson R.T. Ch'ng L.E. Sieff C.A. Livingston D.M. Yao T.P. Genes Dev. 2000; 14: 272-277PubMed Google Scholar). Studies of specific transcription factor pathways provide additional evidence of differences between the functions of CBP and p300. For example, fibroblasts derived from homozygous p300 knockouts are defective for retinoic acid receptor but not CREB signaling (5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Similarly, ribozyme-mediated ablation of p300, but not CBP, blocks the retinoic acid receptor response (6Kawasaki H. Eckner R. Yao T.P. Taira K. Chiu R. Livingston D.M. Yokoyama K.K. Nature. 1998; 393: 284-289Crossref PubMed Scopus (302) Google Scholar). Other differential functions of CBP and p300 have been revealed by their distinct interactions with viral transforming proteins. For example, the Kaposi sarcoma-associated herpesvirus protein vIRF has been reported to be stimulated by CBP and repressed by p300 (7Jayachandra S. Low K.G. Thlick A.E., Yu, J. Ling P.D. Chang Y. Moore P.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11566-11571Crossref PubMed Scopus (87) Google Scholar). Distinct roles for CBP and p300 have also been suggested in the differentiation of muscle and F9 teratocarcinoma cells (8Puri P.L. Sartorelli V. Yang X.J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 9Ugai H. Uchida K. Kawasaki H. Yokoyama K.K. J. Mol. Med. 1999; 77: 481-494Crossref PubMed Scopus (31) Google Scholar). On the other hand, homozygous mutations in CBP and p300 both result in lethality and a similar constellation of phenotypic defects (4Kung A.L. Rebel V.I. Bronson R.T. Ch'ng L.E. Sieff C.A. Livingston D.M. Yao T.P. Genes Dev. 2000; 14: 272-277PubMed Google Scholar, 5Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson R.T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Moreover, CBP/p300 double heterozygotes are invariably lethal, suggesting that functions of CBP and p300 must overlap, at least to some degree. This complexity is not shared by simpler metazoans, such as Drosophila andCaenorhabditis elegans, which express only a single isoform of CBP/p300 (reviewed in Ref. 2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). Although cell cycle-dependent phosphorylation of p300 was reported almost a decade ago (10Yaciuk P. Carter M.C. Pipas J.M. Moran E. Mol. Cell. Biol. 1991; 11: 2116-2124Crossref PubMed Scopus (92) Google Scholar), it is still not entirely clear how phosphorylation regulates CBP/p300 function. In large part, this lack of understanding is because of the fact that the specific phosphorylation sites in CBP/p300 have never been precisely identified. Phosphorylation of p300 and CBP by cyclin E/Cdk2 was reported by Perkins et al. (11Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar) and Ait-Si-Ali et al. (12Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar), respectively. In the case of p300, cyclin E/Cdk2 was shown to negatively regulate coactivator function in a manner that can be blocked by the cyclin-dependent kinase inhibitor, p21. In this model, p21 was proposed to participate in a positive feedback loop, whereby activators such as p53, which depend upon p300 for function, induce p21, which then alleviates the block in p300 action mediated by cyclin E/Cdk2. In contrast, cyclin E/Cdk2 was reported to increase the intrinsic HAT activity of CBP, potentially activating expression of S-phase genes that are repressed in early G1(12Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar). Because the phosphorylation sites in p300 and CBP have not been mapped, however, it may be premature to conclude that the two coactivators are differentially regulated by cyclin-dependent kinases. CBP and p300 both contain a consensus protein kinase A (PKA) site adjacent to their third zinc finger domains, and several groups have proposed that phosphorylation by PKA may contribute to CBP/p300 regulation. For example, Xu et al. (13Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (248) Google Scholar) have argued that phosphorylation of CBP is responsible for the PKA-mediated augmentation of the transcription factor Pit-1. This is an intriguing model because Pit-1 itself cannot be phosphorylated by PKA. Using microinjection assays, these workers demonstrated that the activation of Pit-1 by PKA was lost in the presence of CBP containing a point mutation at the consensus PKA site. This model was not confirmed by Zanger et al. (14Zanger K. Cohen L.E. Hashimoto K. Radovick S. Wondisford F.E. Mol. Endocrinol. 1999; 13: 268-275PubMed Google Scholar), however, and Swope et al. (15Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) have suggested that the PKA-responsive domain in CBP resides near its amino terminus. At this point, the mechanism of PKA activation of CBP/p300 remains enigmatic. Other kinases proposed to regulate CBP/p300 function include calcium/calmodulin (CaM) kinase IV, MAPK, and pp90Rsk. Although several reports suggested that CBP cannot mediate its transcriptional functions in the absence of CaM kinase IV stimulation (16Chawla S. Hardingham G.E. Quinn D.R. Bading H. Science. 1998; 281: 1505-1509Crossref PubMed Scopus (379) Google Scholar, 17Hardingham G.E. Chawla S. Cruzalegui F.H. Bading H. Neuron. 1999; 22: 789-798Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 18Hu S.C. Chrivia J. Ghosh A. Neuron. 1999; 22: 799-808Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), later studies showed that recruitment of CBP by itself was sufficient for transcriptional activation (19Cardinaux J.R. Notis J.C. Zhang Q. Vo N. Craig J.C. Daniel F.M. Brennan R.G. Goodman R.H. Mol. Cell. Biol. 1999; 20: 1546-1552Crossref Scopus (155) Google Scholar, 20Du K. Asahara H. Jhala U.S. Wagner B.L. Montminy M. Mol. Cell. Biol. 2000; 20: 4320-4327Crossref PubMed Scopus (90) Google Scholar). It remains possible, however, that phosphorylation of CBP/p300 by CaM kinase IV could contribute to signaling by augmenting the transcriptional response. Phosphorylation and activation of CBP by MAPK was first reported by Janknecht and Nordheim (21Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar). Activation of MAPK through the Ras pathway by insulin or nerve growth factor was reported to recruit pp90Rsk to the third zinc finger domain of CBP in a manner that prevents the binding of essential CBP effectors such as RNAPII (22Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Interestingly, modulation of CBP by pp90Rsk does not appear to require its catalytic kinase activity. This inhibitory effect of pp90Rsk has not been seen by other investigators, however, so it is possible that it is cell type-specific (23Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1085) Google Scholar). Clearly, the understanding of CBP/p300 regulation by phosphorylation remains a major topic for future study. In addition to their intrinsic acetyltransferase functions, CBP and p300 are known to associate with additional HATs, including P/CAF, SRC-1, and p/CIP. Why so many different HATs are required for transcriptional regulation is unknown, but the answer may lie in the differing preferences of these enzymes for free histones as compared with nucleosomes and their distinct targets within the histone substrates (for review, see Ref. 24Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6623) Google Scholar). Although it has been suggested that the HAT domains in CBP/p300 are highly related to those in P/CAF and GCN5 (25Martinez-Balbas M.A. Bannister A.J. Martin K. Haus-Seuffert P. Meisterernst M. Kouzarides T. EMBO J. 1998; 17: 2886-2893Crossref PubMed Scopus (227) Google Scholar), the primary sequences of these domains are actually quite different. Moreover, these differences are significant enough to allow the development of specific inhibitors of the P/CAF and CBP/p300 enzymatic activities (26Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ait S. Khalil E.M. Vassilev A. Wolffe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; 5: 589-595Abstract Full Text Full Text PDF PubMed Google Scholar). Kraus et al. (27Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar) have shown that the p300-mediated activation of estrogen receptor (ER) function on reconstituted chromatin depends upon the intrinsic acetyltransferase activity of the coactivator, demonstrating that this enzymatic function is essential in the context of chromatin. Because histone acetylation is not required for transcription of naked DNA templates, these results imply that some component of chromatin is the acetylation target. These conclusions are supported by the results of Ludlam et al., 2W. Ludlam, R. H. Goodman, and S. Smolik, submitted for publication. which show that flies containing an acetyltransferase-deficient form of CBP are incapable of activating specific target genes in vivo. Although confirming the importance of the CBP enzymatic function, these studies do not identify the acetylation target. Recent studies have shown that the HAT activity of CBP/p300 is directed toward nucleosomes through interactions with the histone chaperone, RbAp 48 (28Zhang Q. Vo N. Goodman R.H. Mol. Cell. Biol. 2000; 20: 4970-4978Crossref PubMed Scopus (54) Google Scholar). Moreover, Ito et al. (29Ito T. Ikehara T. Nakagawa T. Kraus W.L. Muramatsu M. Genes Dev. 2000; 14: 1899-1907PubMed Google Scholar) have found that histone acetylation by p300 facilitates the transfer of H2A-H2B from nucleosomes to the chaperone protein NAP-1. In this model, the recruitment of p300 and the subsequent histone acetylation follow a chromatin remodeling step mediated by ATP-dependent proteins in the ISWI family. These results are consistent within vivo chromatin immunoprecipitation experiments in yeast showing that the association of SWI/SNF components on the HO promoter is required for the subsequent HAT recruitment (30Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 31Krebs J.E. Kuo M.H. Allis C.D. Peterson C.L. Genes Dev. 1999; 13: 1412-1421Crossref PubMed Scopus (251) Google Scholar). Whether the release of H2A-H2B results from the acetylation of these proteins directly or whether other nucleosomal components are the primary targets of the acetyltransferases remains to be determined. Acetylation of transcription factors (through FAT, factoracetyltransferase activities) by CBP/p300 may provide an equally important mode of regulation. First identified in the context of the tumor suppressor p53 (32Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2177) Google Scholar), acetylation of transcription factors has been increasingly recognized as a mechanism of gene regulation. In some instances, acetylation has clearly been shown to increase the binding of transcription factors to DNA (32Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2177) Google Scholar). In most cases, however, the mechanism of activation is unknown. Recent evidence suggests that coactivator acetyltransferases might also serve to disrupt activator and repressor complexes. For example, Evans and co-workers (33Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar) have shown that the recruitment of p300 to the ligand-activated ER leads to the acetylation of ACTR (an associated acetyltransferase), disruption of the ACTR-p300-ER complex, and the termination of transcription. Another possibility, consistent with the multistep model of transcription proposed by Roeder (reviewed in Refs.34Roeder R.G. Chambon P. Fukasawa T. Kornberg R. Coath C. Transcription Regulation in Eukaryotes. Human Frontiers Science Program, Strausbourg, France1999: 106-121Google Scholar and 35Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), is that CBP/p300-mediated acetylation of the complex may promote the transition from a CBP/p300-dependent to a mediator-dependent stage of transcription (see below). A converse mechanism was proposed by Zhang et al. 3Zhang, Q., Yao, H., Vo, N., and Goodman, R. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97,14323–14328. In these studies, interaction of the histone deacetylase-binding corepressor, CtBP (carboxyl-terminal bindingprotein), to a variety of transcriptional repressors was shown to be blocked by acetylation of the CtBP interaction sites. In this instance, as in the classical histone acetylation model, acetylation is proposed to activate transcription by disrupting protein complexes involved in repression. Paradoxically, acetylation by CBP can also cause transcriptional repression in some systems. For example, in flies, CBP has been shown to inhibit wingless signaling by acetylating the Drosophila homologue of the high mobility group protein, LEF/TCF-1 (36Waltzer L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (326) Google Scholar). Acetylation of a specific residue in LEF/TCF-1 is believed to block the binding of the coactivator β-catenin/Armadillo, one of the intermediates in the wingless signaling pathway. In support of this model, CBP loss-of-function mutants have been found to suppress the effects of an Armadillo mutation. It is somewhat surprising that CBP and p300, which mediate the activities of so many different transcription factors, might be present in the cell at limiting concentrations. Nonetheless, there is considerable evidence that this is the case. Even discounting experiments involving transcription factor overexpression, which would perhaps be expected to exceed the capacity of the endogenous CBP/p300, studies have shown that relatively small decreases in the concentrations of coactivator are deleterious. For example, in the human Rubinstein-Taybi syndrome, loss of a single CBP allele results in severe developmental defects (37Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C. Masuno M. Tommerup N. van Ommen G.J. Goodman R.H. Peters D.J. Bruening M.H. Nature. 1995; 376: 348-351Crossref PubMed Scopus (1024) Google Scholar). The idea that CBP/p300 levels are limiting is also supported by tissue culture experiments, as exemplified by the studies of Hottiger et al. (38Hottiger M.O. Felzien L.K. Nabel G.J. EMBO J. 1998; 17: 3124-3134Crossref PubMed Scopus (127) Google Scholar), which examined the ability of interferon-α (IFN-α) to inhibit tumor necrosis factor-α-stimulated human immunodeficiency virus gene expression. This inhibition was shown to be mediated by competition between STAT-2 (stimulated by IFN-α) and the p65 subunit of NF-κB (stimulated by tumor necrosis factor-α) for a shared binding site within the first zinc finger domain of CBP/p300. It is not certain that two transcription factors must compete for the same binding site to be mutually antagonistic, however. If CBP/p300 levels are truly limiting, it is possible that they could be directed toward specific genes to the exclusion of others. Testing this hypothesis will require the use of experimental paradigms that do not involve the overexpression of exogenous transcription factors. The idea that CBP/p300 contributes to transcriptional synergy is probably best supported by studies of the IFN-β enhanceosome (see below), but other complex promoters have also been shown to contain binding sites for multiple CBP/p300-interacting transcription factors. (Indeed, given the large number of factors that bind CBP/p300, it is difficult to imagine a promoter where this would not be the case.) Nonetheless, although transcriptional synergy through CBP/p300 is an appealing model, it has not been shown conclusively that these coactivators interact with multiple transcription factors simultaneously. In addition, although the recruitment of coactivators to the enhanceosome appears to be required for synergistic activation, tethering CBP/p300 to the promoter through a heterologous DNA-binding domain is not sufficient. As suggested by Merika et al.(39Merika M. Williams A.J. Chen G. Collins T. Thanos D. Mol. Cell. 1998; 1: 277-287Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), the activation domains of the transcription factors the enhanceosome may contribute interactions with factors. CBP/p300 may only participate in a step in the transcriptional It may be equally important for CBP/p300 to be by other factors, such as the complex, for transcription to In support of this Kraus and Kadonaga W.L. Kadonaga J.T. Genes Dev. 1998; PubMed Scopus Google Scholar) have demonstrated that although both the ER and p300 are for transcriptional from chromatin templates, only the ER is required for processes are regulated through the interactions of a large number of complexes. of transcription factors at the promoter the result of these Regulation is by additional components such as and complexes with specific and are complexes that promote the recruitment of coactivators and the RNAPII complex to sites of transcription. In one example, of the enhanceosome recruitment of factors, and to in the IFN-β promoter to interaction between the binding proteins and the RNAPII complex M.G. Lin T. Mol. Cell. 1998; 1: Full Text Full Text PDF PubMed Scopus Google Scholar, T.K. T. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The role of CBP/p300 in this context is to promote the of the and complex to multiple of transcription J. Merika M. N. Chen G. Thanos D. EMBO J. 1999; PubMed Scopus Google Scholar). of CBP/p300 from this complex the of transcription J. K. Thanos D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). CBP/p300 may also participate in IFN-β gene transcription by acetylating its for DNA and disrupting the enhanceosome J. Merika M. N. Chen G. Thanos D. EMBO J. 1999; PubMed Scopus Google N. Merika M. J. K. Chen G. Thanos D. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus (305) Google Scholar, M. J.E. M. Vassilev A. Nakatani Y. Martin B. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Whether actually on the HAT activity of CBP/p300 has not been however. Recent evidence demonstrating an HAT activity in one of the DNA-binding proteins found in the IFN-β suggests that the CBP/p300 HAT function could be H. L. Chiu R. K. Taira K. Nakatani Y. Yokoyama K.K. Nature. 2000; PubMed Scopus Google Scholar). complexes provide the step in the activation to the recruitment of the general transcriptional These and share a of components (reviewed in R.E. Nature. 1999; PubMed Scopus Google Scholar and 35Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The between these complexes and CBP/p300 have not been entirely however. the first of the to be does not contain CBP/p300 and HAT activity H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, M. S. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). with its absence of associated HAT activities and with its to the yeast coactivator functions with activators on naked DNA templates, additional functions with chromatin remain to be In contrast, and transcription has been on chromatin templates, the presence of some components in the with DNA the association or of CBP/p300 other with and complexes P.A. Zhou S. S. W. R. Nature. 1999; PubMed Scopus Google Scholar, C. B.D. V. M. H. P. Nature. 1999; PubMed Scopus Google Scholar). components also additional essential functions with chromatin templates, a for additional interactions for of the complex in this is the role of CBP/p300 in and CBP/p300 transcriptional activators to the complex, or is its role to in a manner that the to function at a subsequent stage of of the regarding the association of CBP/p300 with complexes may from the different to these was a functional H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and identified through their binding to transcription factors C. B.D. V. M. H. P. Nature. 1999; PubMed Scopus Google Scholar). It is that the proteins to and interact with CBP/p300 and the in a mutually This would be consistent with the multistep interaction model proposed by Roeder S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). In this model, transcription factors have the capacity to interact with both CBP/p300 and the but the interactions might be nucleosomal have been by CBP/p300. Nonetheless, it may be premature to conclude that do not contain et al. Y. J. Kornberg Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) have that the yeast interactions with nucleosomes and a that nucleosomal histone at least in yeast does not contain complexes do have intrinsic HAT activity. These studies the of whether the complexes might also contain associated HATs or whether this activity must be by a distinct complex containing CBP/p300. In the step model of transcriptional coactivator HATs such as CBP/p300 are in the and of the transcription how CBP/p300 the for subsequent in the transcriptional remains to be determined. the activation, and termination of CBP/p300 functions will on how cells use transcriptional complexes to mediate specific to cellular We and W. Kraus for protein protein histone acetyltransferase RNA polymerase II holoenzyme protein kinase A protein kinase factor acetyltransferase estrogen receptor interferon-α protein protein

The ability of central glutamatergic synapses to change their strength in response to the intensity of synaptic input, which occurs, for example, in long-term potentiation (LTP), is thought to provide a cellular basis for memory formation and learning. LTP in the CA1 field of the hippocampus requires activation of Ca2+/calmodulin-kinase II (CaM-KII), which phosphorylates Ser-831 in the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-R), and this activation/phosphorylation is thought to be a postsynaptic mechanism in LTP. In this study, we have identified a molecular mechanism by which CaM-KII potentiates AMPA-Rs. Coexpression in HEK-293 cells of activated CaM-KII with GluR1 did not affect the glutamate affinity of the receptor, the kinetics of desensitization and recovery, channel rectification, open probability, or gating. Single-channel recordings identified multiple conductance states for GluR1, and coexpression with CaM-KII or a mutation of Ser-831 to Asp increased the contribution of the higher conductance states. These results indicate that CaM-KII can mediate plasticity at glutamatergic synapses by increasing single-channel conductance of existing functional AMPA-Rs or by recruiting new high-conductance-state AMPA-Rs.

Although opioids are highly effective for the treatment of pain, they are also known to be intensely addictive. There has been a massive research investment in the development of opioid analgesics, resulting in a plethora of compounds with varying affinity and efficacy at all the known opioid receptor subtypes. Although compounds of extremely high potency have been produced, the problem of tolerance to and dependence on these agonists persists. This review centers on the adaptive changes in cellular and synaptic function induced by chronic morphine treatment. The initial steps of opioid action are mediated through the activation of G protein-linked receptors. As is true for all G protein-linked receptors, opioid receptors activate and regulate multiple second messenger pathways associated with effector coupling, receptor trafficking, and nuclear signaling. These events are critical for understanding the early events leading to nonassociative tolerance and dependence. Equally important are associative and network changes that affect neurons that do not have opioid receptors but that are indirectly altered by opioid-sensitive cells. Finally, opioids and other drugs of abuse have some common cellular and anatomical pathways. The characterization of common pathways affected by different drugs, particularly after repeated treatment, is important in the understanding of drug abuse.

MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.