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The Metabotropic GABAB Receptor Directly Interacts with the Activating Transcription Factor 4

The Metabotropic GABAB Receptor Directly Interacts with the Activating Transcription Factor 4 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 45, Issue of November 10, pp. 35185–35191, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Metabotropic GABA Receptor Directly Interacts with the Activating Transcription Factor 4* Received for publication, March 31, 2000, and in revised form, July 28, 2000 Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M002727200 Ralf B. Nehring‡§, Hiroshi P. M. Horikawa§¶i, Oussama El Far§¶, Matthias Kneussel¶, Johann Helmut Brandsta ¨ tter**‡‡, Stefan Stamm§§, Erhard Wischmeyer‡, Heinrich Betz¶, and Andreas Karschin‡ ¶¶ From the ‡Department of Molecular Neurobiology of Signal Transduction, Max Planck Institute for Biophysical Chemistry, 37070 Go ¨ ttingen, the Departments of ¶Neurochemistry and **Neuroanatomy, Max Planck Institute for Brain Research, 60528 Frankfurt, and the §§Research Group for Neuron-specific Splicing, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany G protein-coupled receptors regulate gene expression ognize cis-acting promoter and enhancer elements (5). Among the best studied examples of DNA target motifs in many neu- by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. ronal genes is the octanucleotide cAMP response element In the central nervous system, heteromeric metabo- (CRE) that is bound by transcription factors of the ATF/CREB g-aminobutyric acid type B (GABA tropic ) receptors B family when phosphorylated by protein kinase A upon an in- through adenylyl cyclase regulate cAMP levels, which crease in cellular cAMP levels (6, 7). Inhibitory neurotransmit- may control transcription factor binding to the cAMP ters that lower cytoplasmic cAMP levels are expected to nega- response element. Using yeast-two hybrid screens of rat tively regulate neuronal transcription through CREB- brain libraries, we now demonstrate that GABA recep- dependent mechanisms. Indeed, previous reports on the main tors are engaged in a direct and specific interaction inhibitory neurotransmitter in the central nervous system, with the activating transcription factor 4 (ATF-4), a g-aminobutyric acid (GABA), have shown that in cerebellar member of the cAMP response element-binding protein granule neurons the specific agonist baclofen inhibits forskolin- /ATF family. As confirmed by pull-down assays, ATF-4 initiated CREB-transcriptional programs by lowering cytosolic associates via its conserved basic leucine zipper domain cAMP or Ca levels (8). with the C termini of both GABA receptor (GABA R) 1 B B In the central nervous system, GABA targets to two distinct and GABA R2 at a site which serves to assemble these types of receptors, ligand-gated ionotropic GABA receptors receptor subunits in heterodimeric complexes. Confocal (including GABA receptors) and G protein-linked, metabo- fluorescence microscopy shows that GABA R and ATF-4 tropic GABA receptors (GABA R; Refs. 9 –11), thus mediating are strongly coclustered in the soma and at the den- B B both fast and slow inhibition of excitability at central synapses. dritic membrane surface of both cultured hippocampal In short term signaling, presynaptically located GABA Rs sup- neurons as well as retinal amacrine cells in vivo.In B press neurotransmitter release by inhibiting voltage-sensitive oocyte coexpression assays short term signaling of P, N, and L-type Ca GABA Rs via G proteins was only marginally affected channels (11–14). Postsynaptically, by the presence of the transcription factor, but ATF-4 GABA R stimulation generally causes inhibition of adenylate was moderately stimulated in response to receptor acti- a cyclase via G subunits (15), as well as activation of Kir3 type vation in in vivo reporter assays. Thus, inhibitory potassium channels by liberated Gbg subunits, thereby hyper- metabotropic GABA Rs may regulate activity-depend- B polarizing the postsynaptic membrane (16, 17). Molecularly, ent gene expression via a direct interaction with ATF-4. two major isoforms of the metabotropic receptor, GABA R1 and GABA R2, and various splice variants thereof, have been recently described (18 –25). Their primary amino acid se- Many stimulatory neurotransmitters and hormones in the quences indicate heptahelical membrane topology and are most mammalian central nervous system have been found to cause closely related to the family 3 of G-protein coupled receptors: long term changes in neuronal function, such as differentiation, metabotropic glutamate receptors (mGluR; Refs. 26 and 27), plasticity, and learning (1– 4). These changes generally require 21 the Ca sensing receptor (28), and the vomeronasal receptors agonist-driven activation of cellular signaling cascades, fol- (29, 30). In central neurons GABA R1 and GABA R2 are B B lowed by the induction of transcriptional regulators that rec- widely coexpressed and, a novelty for heptahelical receptors, were found to generate fully functional receptors only when * This work was supported in part by Deutsche Forschungsgemein- linked by their C-terminal tails in a heterodimeric assembly schaft Grants SFB 406, SFB 474, and SFB 269 and by the Fonds der (19 –23). Although the precise functional consequences of this Chemischen Industrie. The costs of publication of this article were association have not yet been deciphered in detail, it is thought defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted The abbreviations used are: CRE, cAMP response element; CREB, TM to the GenBank /EBI Data Bank with accession number(s) AF252627. cAMP response element-binding protein; bZIP, basic leucine zipper domain; GABA, g-aminobutyric acid; GABA § These authors contributed equally to this work. R, metabotropic B-type Supported in part by a Human Frontier Science Program postdoc- GABA receptor; Kir, inwardly rectifying potassium channel; NLS, nu- toral fellowship and by the Novartis Foundation for the Promotion of clear localization signal; SFV, Semliki forest virus; Y2H, yeast-two Science. hybrid; MEKK, mitogen-activated protein kinase/extracellular signal- ‡‡ Supported by a Heisenberg fellowship. regulated kinase kinase kinase; ATF, activating transcription factor; ¶¶ To whom correspondence should be addressed: Fax: 49-551-201- GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, 1688; E-mail: akarsch@gwdg.de. phosphate-buffered saline; EGFP, enhanced green fluorescent protein. This paper is available on line at http://www.jbc.org 35185 This is an Open Access article under the CC BY license. Interaction of GABA 35186 R and ATF-4 0.5% (w/v) Triton X-100). Proteins bound to the beads were eluted by that subunit dimerization promotes proper posttranslational SDS sample buffer and analyzed by Western blotting using anti- processing, membrane targeting, and assembly into specific CREB2/ATF-4 antibodies (1:1000; Santa Cruz Biotechnology). Affinity signaling matrices in subcellular neuronal specializations (31). purification of native GABA R1 from the solubilized P2 fraction was By means of yeast-two-hybrid (Y2H) interaction cloning, bio- performed using an aliquot of the P2 fraction. The P2 (8 mg of protein) chemical, and functional reporter assays, as well as immuno- fraction was solubilized for 1 h with 1.5% Triton X-100 in a 3-ml final cytochemistry, we now provide evidence that metabotropic volume of Tris-buffered saline (25 mM Tris, pH 7.4, 150 mM NaCl, and protease inhibitors). After ultracentrifugation at 100,000 3 g for1h, GABA Rs are also capable of directly interacting with tran- the supernatant was incubated for 5 h with 30 ml of glutathione beads scription factors and thus may utilize a mechanism for tran- preloaded with either GST or GST-ATF-4 (150 mg). Bound material was scriptional regulation unique to membrane receptors. Our mu- recovered from glutathione beads after washing four times with Tris- tational analysis indicates that GABA R bind to ATF-4, a buffered saline containing 0.2% (w/v) Triton X-100 and one wash with transcription factor of the leucine zipper ATF/CREB family, via Tris-buffered saline alone. Bound proteins were then eluted with SDS their C-terminal leucine zipper motifs, which in vivo may result sample buffer. Proteins were analyzed by Western blotting using a polyclonal goat anti-GABA R1 antibody (Santa Cruz Biotechnology). in the regulation of gene transcription upon stimulation. B Miscellaneous Methods—SDS-polyacrylamide gel electrophoresis EXPERIMENTAL PROCEDURES was performed on 6%, 10%, or 12% polyacrylamide gels. For Western Yeast Two-hybrid Screening—Two independent Y2H assays were blot analysis, proteins were transferred to nitrocellulose membranes used in parallel. Both the MATCHMAKER II (CLONTECH) and the (Schleicher & Schuell). First antibodies were overlaid with goat anti- LexA (OriGene Technologies) systems were used to screen rat brain rabbit or anti-mouse horseradish peroxidase-coupled secondary anti- cDNA libraries constructed with the activation domain vectors pAD- bodies, and chemiluminescence was detected using the Pico detection GAL4 and pJG4 –5, respectively, using amino acids 848 –960 of the kit (Pierce). C-terminal coding region of GABA R1a (18) as bait. C-terminal baits In Vivo Reporting Systems —The trans PathDetect reporting sys- were amplified by polymerase chain reaction from a rat brain library tem (Stratagene) was used to test if ATF-4 was involved in activating and inserted into the DNA-binding domain vector pGBT9 and the transcriptional expression upon receptor stimulation. For all experi- galactose-inducible vector pGilda, respectively. Screening with the ments HEK293-GBR cells were used that had been stably tranfected pGBT9 and pGilda bait yielded colonies that grew on the corresponding with GABA R1 and GABA R2 subunits. A fusion transactivator pro- B B selection plates complemented with 10 mM 3-aminotriazole for the tein between ATF-4 (or constitutively MEKK-activated Jun) and the MATCHMAKER system and were positive in the b-galactosidase assay. GAL4 DNA binding domain was constructed and transfected (pFA- Isolated plasmids were sequenced on both strands using the ABI ATF-4; 50 ng) into HEK293-GBR cells together with vectors carrying PRISM sequenase dye terminator kit on an automatic sequencer. For the luciferase reporter gene under the control of the GAL4 promoter analysis of the interaction site, GABA R1 and ATF-4 deletions were (pFR-Luc; 1 mg) and a vector expressing b-galactosidase (50 ng). 24 h generated by polymerase chain reaction with specific oligonucleotides after transfection, cells were treated with serum-free medium, and 18 h and subcloned into pGBT9 and pAD-GAL4. Yeast strains HF7c and later lysed with 500 ml of lysis buffer and the supernatant centrifuged EGY 48 were cotransformed with 100 ng each of bait and prey vector, to remove cell debris. Luciferase activity was measured with a lumi- streaked out on agar plates lacking tryptophan, leucine, and histidine nometer (Luciferase Reporter Gene Assay, Roche Diagnostics). (MATCHMAKER), and also lacking uracil (LexA). Colony growth/acti- Electrophysiology—For expression in Xenopus laevis oocytes, capped vation of the HIS3 and LEU reporter genes, respectively, as well as run-off poly(A ) cRNA transcripts were synthesized from GABA R1a, b-galactosidase activity were controlled after 4 days. GABA R2, ATF-4, and Kir3.1/3.2 concatemers (32) and ;3 ng of each Preparation of Brain Homogenates —Rat cerebral cortices were ho- injected in defolliculated oocytes. Oocytes were incubated at 19 °C in mogenized in a Teflon glass Potter homogenizer with 12 strokes at 900 ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl ,1mM CaCl ,5mM 2 2 rpm in 20 ml of ice-cold 0.32 M sucrose, 4 mM HEPES/NaOH, pH 7.3, HEPES, pH 7.4 -7.5), supplemented with 100 mg/ml gentamicin and 2.5 containing Complete and a protease inhibitor mixture (Roche Diagnos- mM sodium pyruvate, and assayed 72 h after injection. Two-electrode tics). The homogenate was centrifuged for 10 min at 800 3 g in a Sorvall voltage-clamp measurements were performed with a Turbo Tec-10 C SS-34 rotor (DuPont). The resulting pellet was used as crude nuclear amplifier (npi) and sampled through an EPC9 (Heka Electronics) inter- fraction (P1). The supernatant was recovered and spun at 27,000 3 g for face using Pulse/Pulsefit software (Heka). Oocytes were placed in a another 30 min. The resulting pellet (P2) was resuspended in 3 ml of small volume perfusion chamber and bathed with ND96 or “high K ” homogenization buffer and frozen until needed. solution (96 mM KCl, 2 mM NaCl, 1 mM MgCl ,1mM CaCl ,5mM 2 2 Bacterial Recombinant Fusion Proteins —Glutathione S-transferase HEPES, pH 7.4 –7.5). (GST) fusion proteins of GABA R (GST-GBR1 and GST-GBR2) and Neuronal Expression via Semliki Forest Viruses (SFV) and Immuno- synaptoporin tail regions (amino acids 198 –265) were constructed in cytochemistry —Recombinant SFV harboring ATF-4 were engineered pGEX-5X-1 (Amersham Pharmacia Biotech) using specific EcoRI- and and processed as described previously (33). In brief, the cDNA of the SalI-flanked oligonucleotides. Full-length ATF-4 was fused to the C N-terminally EGFP-tagged fusion protein ATF-4-EGFP was subcloned terminus of GST in pGEX-5X-1, and maltose-binding protein (MBP- into pSFV1 (Life Technologies, Inc.). After linearization with SpeI, ATF-4) in pMAL-c2 (New England Biolabs) using EcoRI and XhoI sites, cDNA was in vitro transcribed using SP6 RNA polymerase (Roche and electroporated into Epicurian Escherichia coli BL21 (Stratagene). Molecular Biochemicals). BHK21 cells were transfected by electropora- Expression of fusion protein was induced by 1 mM isopropyl-1-thio-b-D- tion (400 V, 975 microfarads) with a mixture of 10 mg of pSFV/ATF-4- galactopyranoside for 3–5 h. Cells were broken in a French press in EGFP and pSFV-helper2, respectively. After 24 h supernatant was PBS, and soluble protein fractions were recovered in the supernatant collected and stored in 450-ml aliquots at 280 °C. Prior to treatment of after centrifugation at 100,000 3 g for 1 h and kept frozen until needed. neuronal cultures, aliquots of virus were activated by 100 ml of chymo- MBP-ATF-4 Binding to GST Fusion Proteins—Glutathione beads (30 trypsin (2 mg/ml). Primary cultures of hippocampal neurons were pre- ml) were loaded by incubation for1hat4 °CinPBS with 150 mgofGST pared from 1-day-old rats as described previously (34). Hippocampal and the fusion proteins between GST and the C termini of synaptoporin, neurons were cultured in Neurobasal A medium supplemented with GBR1 and GBR2. An extract (100 ml) of bacterially expressed MBP- B27 (Life Technologies, Inc.) in 12-well plates (2 ml in each well) for 14 ATF-4 was then added to preloaded beads in binding buffer (PBS, 0.2% days. Half of the medium was removed from each well and stored at Triton X-100, and Complete) and rotated at 4 °C for 3 h. Pelleted beads 37 °C. For infection, 20 –50 ml of activated virus was added per well. were washed three times with binding buffer and once with PBS. Bound After incubation for2hat37 °C,the virus-containing medium was proteins were eluted using SDS sample buffer and separated on a 10% replaced with stored aliquots. Expression of ATF-4-EGFP was observed SDS-polyacrylamide gel. To control for MBP-ATF-4 binding, samples 12–14 h after infection. were subjected to Western blotting with rabbit anti-MBP antibodies For immunostaining, hippocampal neurons were fixed with 2% (w/v) (1:10,000; New England Biolabs). paraformaldehyde, 0.1% (w/v) Triton X-100 and blocked with 2% (v/v) Pull-down Assays—ATF-4 pull-downs from P1 brain extract were normal goat serum. Subsequently, neurons were incubated overnight achieved by mixing 100 ml of GST or GST-GBR1 bacterial extracts with with guinea pig anti-GABA R1 (1:1000; PharMingen) and rat anti- 10 mg of brain P1 Triton extract in 10 ml of 10 mM HEPES/NaOH, pH synaptophysin (1:1000; a gift of R. Jahn, Go ¨ ttingen) antibodies, respec- 7.3, containing 0.5% (w/v) Triton X-100 for2hat4 °C. After incubation, tively. After washing with PBS, cells were incubated 1 h with Cy3- 50 ml of glutathione beads were added and the mixture was incubated conjugated IgG (1:1000; Jackson Immunoresearch Laboratories). for an additional 2 h. Glutathione beads were then recovered by cen- Coverslips were washed again with PBS, mounted on slides, and ana- trifugation and washed three times vigorously with PBST (PBS with lyzed on a confocal LSM410 microscope (Zeiss). Interaction of GABA R and ATF-4 35187 FIG.1. Amino acid sequence alignment of rat and mouse ATF-4. Shown are the leucine zipper I and II motifs, the putative mitogen-activated protein kinase phosphorylation site, and the basic DNA binding domain. The rATF-4 sequence has been deposited to GenBanky under accession no. AF252627. Retina Preparation and Immunocytochemistry—Adult albino rats were anesthetized deeply with halothane and decapitated. Eyes were enucleated and opened along the ora serrata, and the posterior eyecups with the retinae attached were immersion-fixed for 15–30 min in 4% (w/v) paraformaldehyde in 0.1 M PB, pH 7.4. After dissection retinae were cryoprotected in 10% (w/v), 20% (w/v) sucrose in PB for 1 h each and in 30% (w/v) sucrose in PB overnight at 4 °C. Pieces of retinae were mounted in freezing medium (Reichert-Jung, Bensheim, Germany), sectioned vertically at 12-mm thickness on a cryostat, and collected on slides. For double-labeling experiments, guinea pig anti-GABA R1 (1: 1000; PharMingen) and rabbit anti-CREB2/ATF-4 antibodies (1:1000; Santa Cruz Biotechnology) were used and visualized by red and green fluorescence secondary antibodies, goat anti-rabbit IgG, and goat anti- guinea-pig IgG (Alexay 594, Alexay 488; 1:500; Molecular Probes). Sections were examined by confocal laser-scanning microscopy (Leica DM IRBE; Leica Microsystems, Heidelberg, Germany) using a 363 objective and special filter settings (Leica TCS SP). FIG.2. GAL-4-based Y2H analysis of the interaction between RESULTS GABA R1 and ATF-4. A, deletion constructs of GABA R1 (amino acid B B Y2H Assay—Using the Y2H system (35), we sought to isolate region on the left) were used as baits for binding ATF-4 as prey. The HIS3 marker was used as reporter in the Y2H assay. B, the equivalent candidate proteins that directly interact with and modulate the analysis is shown for deletion constructs of rATF-4 used as prey for signaling function of GABA receptors. Therefore, the complete binding the C terminus of GABA R1. Functional sites depicted in Fig. C-terminal intracellular region of GABA R1a was initially 1are boxed in this schematic representation of ATF-4. C, helical used as bait to screen rat brain cDNA libraries. Two independ- wheel diagrams of the putative leucine zipper-based interface between GABA R1 and rATF-4. The view is from the N termini starting from ent screenings of ;2 3 10 recombinants resulted in the isola- S887 (position a in GABA R1a) and Q299 (a9 in rATF-4). Heptad tion of 27 and 180, respectively, positive clones, the open read- B positions are labeled a– g (a9–g9). ing frames of which all encoded regions of the same polypeptide. Data base analysis indicated very high similarity to the mouse transcription factor mATF-4 (36), also known as were constructed and tested for complementation in the Y2H C/ATF (37) or mTR67 (38). The complete open reading frame of assay. These experiments showed that deletion constructs in the rat orthologue (rATF-4) is shown in Fig. 1. rATF-4 is 347 the GABA R1 bait, removing partial sequences from the C amino acids in length and shares 94% and 86% amino acid terminus, allow binding of rATF-4 (Fig. 2A) until a leucine at identity with mouse ATF-4 and the human ATF-4 (hCREB2/ amino acid position 915 (Leu-915) is removed (DQ914) or ex- TAXREB67; Refs. 39 and 40), respectively. At the C terminus, changed by a glycine (L915G) or serine (L915S) residue (data rATF-4 harbors a conserved basic leucine zipper (bZIP) dimer- not shown in the illustration). In contrast, replacement of Gln- ization motif that binds CRE and is present in all CREB/ATF 916 by an alanine (Q916A) did not disturb the interaction. proteins. In addition, rATF-4 contains a second heptad repeat Further restriction analysis on the 59 end of GABA R1a as- of leucines between amino acids 89 and 124 near the N termi- signs the region of interaction to amino acids 887–915. Inter- nus, and a potential phosphorylation site for mitogen-activated estingly, this domain has been recently mapped to likely par- protein kinase (amino acid position 164). ticipate in the obligate assembly of GABA R1 and GABA R2 B B We performed control experiments (i) with bait or prey vec- subunits into heteromeric receptor complexes (23, 41), suggest- tors missing, (ii) using empty vectors, (iii) using vectors that ing a bifunctional role of this site. expressed unrelated proteins such as pRHFM1 encoding the Conversely, deletion constructs in rATF-4 demonstrated that Drosophila bicoid protein homeodomain, and (iv) using vectors the first leucine zipper an the putative mitogen-activated pro- that expressed other proteins harboring a bZIP domain, e.g. tein kinase site of rATF-4 were dispensable for binding, CREB. All these controls were negative, excluding autoactiva- whereas the C-terminal leucine zipper (amino acids 301–337) tion and corroborating the specificity of the interaction between was required for association with GABA R1 (Fig. 2B). When GABA R1 and rATF-4. For a detailed mapping of the interac- described in terms of the heptad patterns seen in a helical tion domains, deletion mutants of both GABA R1 and ATF-4 wheel diagram, the interaction sites between the C termini of B Interaction of GABA 35188 R and ATF-4 FIG.4. Expression in Xenopus oocytes and in vivo luciferase reporter assay. A, expression of GABA R1, GABA R2, ATF-4, and B B FIG.3. Interaction of GABA Rs and ATF-4 in vitro. A, bacterially Kir3.1/3.2 concatemeric channels in Xenopus oocytes elicits basal and expressed GST and GST fusion proteins (GST-Por, fusion with the agonist-dependent (as indicated by black bars)K currents in the C-terminal tail of the synaptic vesicle protein synaptoporin; GST-GBR1 absence (top left trace) and presence of injected ATF-4 antibodies (bot- and GST-GBR2, fusion proteins of the C-terminal tails of GABA R1a tom left trace). High K (96 mM, arrowheads) and 10 mM baclofen (black and R2) were immobilized on glutathione-Sepharose and then incu- bars) were applied to the bath and oocytes voltage-clamped at -80 mV. bated with recombinant MBP-ATF-4. B, GST-GBR1 or GST-ATF-4 Dashed lines indicate zero-current levels, scale bar represents 2 mA and were used to test the binding of ATF-4 (left panel) and native GBR1 10 s. The right panel shows a bar graph summary of Kir3.1/3.2 inward (right panel) present in a Triton X-100 extract from rat brain. Bound currents at -80 mV in the presence (black bars) and absence (white bars) material was eluted using SDS sample buffer, separated by SDS-PAGE, of overexpressed ATF-4 and injected anti-ATF-4 antibodies. B, in vivo and immunoblotted with anti-MBP (A), anti-ATF-4 (B, left), or anti- luciferase trans-reporter assay performed on HEK293 cells stably GBR1 (B, right). transformed with GABA R1 and GABA R2. Fusion transactivator pro- B B teins between the GAL4 DNA binding domain and ATF-4 and Jun (activated with MEKK), respectively, were transfected together with ATF-4 and GABA R1 conform with good approximation to the luciferase reporter gene under the control of the GAL4 promoter. classic coiled-coil structures (Fig. 2C). In the bZIP domain of Luciferase activity was measured in the presence or absence of 10 mM baclofen. rATF-4, the periodic array of leucines at every seventh position likely interdigitates with that of a matching helix formed by the GABA of rATF-4, was detected exclusively in the GST-GBR1, but not R1 C terminus to form a zipper-like structure. It has been suggested earlier that bZIP domains not only mediate in the GST eluates (Fig. 3B, left). In parallel, we showed that association between transcription factors prior to DNA binding ATF-4 can bind to native GABA R1 by performing similar (42), but also form coiled-coil structures from up to four helices pull-down experiments using immobilized GST or GST-ATF-4 with various other proteins (43). Together with the array of and Triton X-100 extract from crude P2 rat brain fraction. leucines at position d in GABA R1 and d9 in ATF-4, several Bound material was eluted and analyzed by Western blotting using anti-GABA R1 antibodies. A 130-kDa protein was de- features are consistent with such an interaction: (i) the b-branched amino acids valine, isoleucine (and alanine) occur tected exclusively in the GST-ATF-4, but not in the GST elu- at the alternate hydrophobic positions a and a9; (ii) there are ates (Fig. 3C). highly conserved breaks at these positions caused by polar Electrophysiology and in Vivo Reporting System—In a fur- asparagines; and (iii) the amino acids preceding the alternate ther series of experiments with heterologously expressed pro- juxtaposed hydrophobic residue and following the leucine of the teins, we sought to reveal a functional consequence of the next heptad are often oppositely charged in both proteins to interaction between GABA R and ATF-4. First, we investi- allow electrostatic interactions (38). Consistent with our pull- gated whether cytosolic ATF-4, when binding to the dimeric down experiments (see below), helical wheel representation of receptor, might provide a negative regulator of receptor func- GABA R2, but not the C-terminal splice variant GABA R1d tion in that it interferes with G protein binding and thus classic B B short term signaling. This putative role of ATF-4 on G protein (24), which misses the leucine zipper, does also reveal comple- mentarity to ATF-4 as required for stable dimerization. signaling of the receptors was studied in Xenopus oocytes under Biochemical Assays—To verify the interaction between two-electrode voltage-clamp. Coinjection at a 1:1:1 ratio of ATF-4 and GABA R1a by an independent assay, we evaluated cRNAs for GABA R1, GABA R2, and Kir3.1/3.2 concatemers B B B as target channels for receptor signaling resulted in the expres- binding of the respective bacterially expressed fusion proteins. In addition, we have tested the binding of C-terminal regions of sion of large basal and baclofen-induced inwardly rectifying K GABA currents as described previously (17). Two days after injection, R2 and the unrelated synaptic protein synaptoporin (Por). GST and the GST-fusion proteins GST-GBR1, GST- Kir3 current amplitudes activated by baclofen (10 mM) aver- GBR2, and GST-Por were immobilized on glutathione beads aged 26.0 6 4.5 mA(n 5 7) at 280 mV in the presence of 96 mM and incubated with either MBP or MBP-ATF-4 bacterial ly- external K (Fig. 4A). To test for the role of endogenous ATF-4, sates. After washing the resin, bound material was eluted and anti-ATF-4 antibodies were injected 1 h prior to the experiment analyzed by Western blotting using anti-MBP antibody. MBP- at a dilution of 1:100. In the presence of antibody, the activa- ATF-4, but not MBP (data not shown), specifically interacted tion kinetics of baclofen-induced Kir currents (as determined with both immobilized GST-GBR1 and GST-GBR2, but not from responses to hyperpolarizing voltage steps) was un- with GST or GST-Por (Fig. 3A). changed, but mean amplitudes were slightly increased by sta- For demonstrating that GABA R1 is capable to interact with tistically significant 18% (p , 0.05). Similarly, when ATF-4 was overexpressed under otherwise unchanged recording con- rATF-4 present in rat brain, we performed pull-down experi- ments with immobilized GST or GST-GBR1 using crude P1 rat ditions, ligand-activated currents in the presence of antibody brain extracts. Triton X-100 extracts from a crude rat brain (25.8 6 2.4 mA) were elevated by 12% compared with control nuclear fraction was incubated with GST or GST-GBR1, and conditions (25.1 6 2.1 mA, n 5 7), indicating that the presence protein binding to the fusion proteins was recovered by adding of intrinsic or exogenously expressed ATF-4 plays only a mar- glutathione beads. Bound material was eluted and analyzed by ginal role in receptor-stimulated G protein activation. Western blotting using anti-rATF-4 antibodies. A doublet of Next, we employed the PathDetect luciferase reporting sys- 95–100-kDa proteins, corresponding most probably to a dimer tem to investigate the in vivo consequences of the GABA R/ B Interaction of GABA R and ATF-4 35189 FIG.5. Colocalization of ATF-4 and GABA R1a in hippocampal neurons. Confocal fluorescence micrographs of primary hippocampal cells immunostained with anti-GABA R1 antibodies and detected with Cy3-labeled secondary antibody (A). B, EGFP-tagged ATF-4 expressed through a recombinant Semliki forest virus. C, fluorescence overlay in the detail figure of regions boxed in A and B shows complete cocluster- ing of ATF-4 and GABA R1 in the dendritic membrane. D, fluorescence overlay of ATF-4-EGFP (green) and antibody-labeled synaptophysin (Syn, Cy3 signal in red). Scale bars,20 mm(A and B), 10 mm(C), and 7 mm(D). ATF-4 interaction on transcriptional activation in HEK293 cells that were engineered to stably express GABA R1 and GABA R2. In the trans-reporting assay, the GAL4 DNA bind- ing domain was joined to ATF-4 and the construct cotrans- fected with a GAL4-driven luciferase reporter plasmid. Cells were assayed after 18 h in serum-free medium. Although re- sults varied considerably between different wells, we found that stimulation with 50 mM baclofen for 1 h increased lucifer- ase levels to 144 6 72% (n 5 10) compared with unstimulated cells (Fig. 4B), which is a statistically significant increase (Stu- dent’s t test; p , 0.05). In another experiment, the GAL4 DNA binding domain was fused to c-Jun and stimulated with con- stitutively activate MEKK. Under these conditions stimulation with baclofen significantly decreased luciferase levels to 56 6 16% (Fig. 4B). The precise mechanisms underlying this differ- ential receptor signaling are presently not understood, but are in accordance with previous findings, if we assume that endog- enous ATF-4 is also present in HEK293 cells. ATF-4 has been FIG.6. Colocalization of GABA R1 and ATF-4 in an amacrine found to suppress the transcriptional action of Jun family cell of the rat retina. Confocal laser-scanning micrographs of a trans- members (44), but is a transcriptional activator by itself (Ref. verse section of rat retina showing an amacrine cell in the inner nuclear 45; see “Discussion”). layer (INL) coimmunostained for GABA R1 (green) and ATF-4 (red). In Neuronal Expression via SFV—The cellular expression of the merged panel below, coexpression of the two proteins is clearly visible in the cell’s cytoplasm and along its processes in the inner GABA R1 and ATF-4 in vivo was first analyzed in low density plexiform layer (IPL; yellow-orange). Scale bar,10 mm. rat hippocampal cultures by immunofluorescence. Primary cul- tures of rat hippocampal neurons were infected with an SFV vector containing EGFP-tagged ATF-4 (ATF-4-EGFP) and ex- prominent baclofen-induced inwardly rectifying K currents pression evaluated 12–18 h after infection. In confocal images (data not shown). After staining with Cy3-labeled secondary ATF-4 was found in the nucleus and diffusely in the cytoplasm, antibodies, GABA R1 antibodies gave rise to a punctate mem- but primarily clustered at specific sites in the outer membranes brane pattern of dendritic immunoreactivity that exactly colo- of hippocampal cell somata and along dendrites (Fig. 5B). Un- calized with coexpressed ATF-4 (Fig. 5, A and C). A similar der whole cell patch-clamp conditions, all hippocampal neurons pattern of GABA R immunoreactivity was obtained in hip- cultured from this age displayed GABA Rs as revealed by pocampal cells that were not infected with ATF-4 SFV, ensur- B Interaction of GABA 35190 R and ATF-4 ing that overexpression of ATF-4 did not affect receptor only after agonist-driven release. So far, the functional assays distribution. performed in our study do raise several unsolved questions on In many cases these puncta were not congruent with synap- the stimuli and targets of cytosolic ATF-4. Although trans- tic regions, as indicated by the differential distribution of the fected COS cells and primary neurons in our experiments were presynaptic terminal marker synaptophysin (Fig. 5D) or glu- depleted overnight of serum and other extracellular stimuli, tamic acid decarboxylase that shows inhibitory GABAergic ter- prominent ATF-4 signals often remained in the nuclei. This minals (data not shown). Our findings suggest that GABA R1 may have masked a pronounced, visually detectable transloca- and ATF-4 are clustered predominantly at extrasynaptic sites tion signal of ATF-4 upon receptor stimulation with baclofen. in neuronal cells. As an alternative explanation, ligand binding to the receptor Colocalization in the Retina—For the mammalian retina, it may be insufficient to fully activate ATF-4 as a transcriptional has been shown that GABA Rs are strongly expressed in am- protein, but require a coincident stimulus through another acrine cells both pre- and postsynaptically (46). We therefore signaling protein of the receptor matrix. investigated whether GABA Rs and ATF-4 would indeed co- When using the trans luciferase reporter assay, a moderate cluster at distinct subcellular sites in a native central nervous rise in transcriptional activity via ATF-4 was seen upon recep- system neuron. Fig. 6 depicts a wide field amacrine cell with tor stimulation. This trend was supported by a cis reporting the cell body located in the inner nuclear layer and its processes assay in HEK-GBR1 stable cells, in which CRE-driven lucifer- stratifying in the inner plexiform layer close to the inner nu- ase expression was enhanced by ;20% by baclofen in the pres- clear layer. Immunocytochemical double-labeling experiments ence of 10 ng/ml pertussis toxin (data not shown). In this assay with anti-GABA R and anti-ATF-4 antibodies revealed the B gene expression was reduced by .30% in the absence of per- striking overlap of GABA R immunoreactivity (green) and tussis toxin which disrupts receptor stimulation of G proteins. ATF-4 immunoreactivity (red) both in the cytoplasmic compart- This indicates that, even under experimental suppression of G ment of the amacrine cell and also along its processes and protein-mediated GABA R signaling, ATF-4 may be involved terminal arborizations, suggesting a strong functional relation- in CRE-mediated stimulation of gene expression independent ship between these two proteins in selected neurons. of G proteins. Yet, this action is far from being understood in detail. Similar to other proteins of the CREB/ATF family, ATF- DISCUSSION 4/CREB2 is known to bind to the transcriptional enhancer Here we demonstrate for the first time a tight and direct motif CRE as homo- or heterodimers in conjunction with c-Jun, interaction between a heptahelical neurotransmitter receptor but also together with TATA-binding protein, TFIIB, RAP30 and a soluble transcription factor (ATF-4). We show that the subunit of TFIIF, or the coactivator CREB-binding protein bZIP domain of ATF-4 associates with the GABA R C termini CBP/p300 (38, 45, 52). It has been reported that ATF-4, like in a coiled coil-confirmation, which is common, e.g., among CREB, can act as a transcriptional activator (45), but under a structural proteins and contains between two and four helices. variety of experimental conditions significantly represses CRE- Given that the site at which ATF-4 interacts with both GAB- dependent transcription (39, 44). This bifunctional role may be R1 and likely GABA R2 overlaps with the putative interac- B B explained by the displacement of other CREB activator/coacti- tion site between the two subunits (23, 41), it may be hypoth- vator proteins from the CRE promoter site by high amounts of esized that, at least temporarily, triple-helix structures exist. It ATF-4 (squelching), resulting in inhibition of transcriptional is currently thought that individually neither of the two recep- activation. A repressive action of ATF-4 orthologues on tor subtypes is expressed and transported with high efficiency CREB1-mediated transcription in Aplysia, Drosophila, and ro- to the outer membrane, because of homodimer instability (41). dents (53) has been interpreted to impede synaptic plasticity Instead, the majority of native GABA Rs are likely to exist as and affect spatial and social learning (54, 55). Based on its heterodimers between GABA R1 and GABA R2 (19 –23) with B B repressive character in the nervous system and in analogy to specific electrostatic interactions in their C termini giving rise the function of tumor suppressor genes, CREB2/ATF-4 has to parallel coiled-coil a-helices. Our assays convincingly been suggested to act as a “memory suppressor gene” that showed that ATF-4 is tightly linked to GABA Rs expressed in decreases synaptic strength or removes inhibitory constraints neuronal membranes. Similarly, overlapping edge fluorescence of long term memory storage (56, 57). of ATF-4 and GABA R1 immunoreactivity was observed by Heptahelical receptors are commonly thought to signal pri- confocal microscopy after cotransfection into COS-7 cells (data marily through coupling to G proteins. Only recently, however, not shown). In COS-7 cells that lack GABA R1 and GABA R2, B B they have been found to interact with a growing number of ATF-4 was not seen at the plasma membrane, but only diffuse membrane and cytosolic proteins, including proteins that func- cytoplasmic staining was found, suggesting that GABA Rs tion in signal termination (58), synaptic targeting (59), mito- play a role in recruiting ATF-4 to the outer plasma membrane. genic signaling (60, 61), and translational regulation (62, 63). The notion that in neurons transcription factors are localized in With the direct interaction of metabotropic GABA R and the dendrites and may be retrogradely transported to the nucleus ubiquitously expressed transcription factor ATF-4 (39) demon- has emerged only recently (47). One of the possible cellular strated here, a novel alternative mechanism by which tran- consequences of the documented GABA R/ATF-4 interaction scriptional regulation important for long term memory forma- would be that agonist stimulation of the receptor at the outer tion (64) may be initiated at inhibitory synapses of the surface membrane releases ATF-4, which then translocates mammalian central nervous system, is emerging. into the nucleus to increase the nuclear pool of this transacti- vating protein. Like other transcription factors, ATF-4 is likely Acknowledgments—We are grateful to Drs. B. Bettler and F. Do ¨ ring imported across the nuclear membrane by shuttling proteins for continuous intellectual and experimental support as well as D. that recognize nuclear localization signals (NLS; Refs. 48 and Reuter, A. Niehuis, D. Magalei, A. Hildebrand, and O. Dick for excellent technical assistance. We also thank I. Herford and Dr. C. Rosenmund 49). 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The Metabotropic GABAB Receptor Directly Interacts with the Activating Transcription Factor 4

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 45, Issue of November 10, pp. 35185–35191, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Metabotropic GABA Receptor Directly Interacts with the Activating Transcription Factor 4* Received for publication, March 31, 2000, and in revised form, July 28, 2000 Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M002727200 Ralf B. Nehring‡§, Hiroshi P. M. Horikawa§¶i, Oussama El Far§¶, Matthias Kneussel¶, Johann Helmut Brandsta ¨ tter**‡‡, Stefan Stamm§§, Erhard Wischmeyer‡, Heinrich Betz¶, and Andreas Karschin‡ ¶¶ From the ‡Department of Molecular Neurobiology of Signal Transduction, Max Planck Institute for Biophysical Chemistry, 37070 Go ¨ ttingen, the Departments of ¶Neurochemistry and **Neuroanatomy, Max Planck Institute for Brain Research, 60528 Frankfurt, and the §§Research Group for Neuron-specific Splicing, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany G protein-coupled receptors regulate gene expression ognize cis-acting promoter and enhancer elements (5). Among the best studied examples of DNA target motifs in many neu- by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. ronal genes is the octanucleotide cAMP response element In the central nervous system, heteromeric metabo- (CRE) that is bound by transcription factors of the ATF/CREB g-aminobutyric acid type B (GABA tropic ) receptors B family when phosphorylated by protein kinase A upon an in- through adenylyl cyclase regulate cAMP levels, which crease in cellular cAMP levels (6, 7). Inhibitory neurotransmit- may control transcription factor binding to the cAMP ters that lower cytoplasmic cAMP levels are expected to nega- response element. Using yeast-two hybrid screens of rat tively regulate neuronal transcription through CREB- brain libraries, we now demonstrate that GABA recep- dependent mechanisms. Indeed, previous reports on the main tors are engaged in a direct and specific interaction inhibitory neurotransmitter in the central nervous system, with the activating transcription factor 4 (ATF-4), a g-aminobutyric acid (GABA), have shown that in cerebellar member of the cAMP response element-binding protein granule neurons the specific agonist baclofen inhibits forskolin- /ATF family. As confirmed by pull-down assays, ATF-4 initiated CREB-transcriptional programs by lowering cytosolic associates via its conserved basic leucine zipper domain cAMP or Ca levels (8). with the C termini of both GABA receptor (GABA R) 1 B B In the central nervous system, GABA targets to two distinct and GABA R2 at a site which serves to assemble these types of receptors, ligand-gated ionotropic GABA receptors receptor subunits in heterodimeric complexes. Confocal (including GABA receptors) and G protein-linked, metabo- fluorescence microscopy shows that GABA R and ATF-4 tropic GABA receptors (GABA R; Refs. 9 –11), thus mediating are strongly coclustered in the soma and at the den- B B both fast and slow inhibition of excitability at central synapses. dritic membrane surface of both cultured hippocampal In short term signaling, presynaptically located GABA Rs sup- neurons as well as retinal amacrine cells in vivo.In B press neurotransmitter release by inhibiting voltage-sensitive oocyte coexpression assays short term signaling of P, N, and L-type Ca GABA Rs via G proteins was only marginally affected channels (11–14). Postsynaptically, by the presence of the transcription factor, but ATF-4 GABA R stimulation generally causes inhibition of adenylate was moderately stimulated in response to receptor acti- a cyclase via G subunits (15), as well as activation of Kir3 type vation in in vivo reporter assays. Thus, inhibitory potassium channels by liberated Gbg subunits, thereby hyper- metabotropic GABA Rs may regulate activity-depend- B polarizing the postsynaptic membrane (16, 17). Molecularly, ent gene expression via a direct interaction with ATF-4. two major isoforms of the metabotropic receptor, GABA R1 and GABA R2, and various splice variants thereof, have been recently described (18 –25). Their primary amino acid se- Many stimulatory neurotransmitters and hormones in the quences indicate heptahelical membrane topology and are most mammalian central nervous system have been found to cause closely related to the family 3 of G-protein coupled receptors: long term changes in neuronal function, such as differentiation, metabotropic glutamate receptors (mGluR; Refs. 26 and 27), plasticity, and learning (1– 4). These changes generally require 21 the Ca sensing receptor (28), and the vomeronasal receptors agonist-driven activation of cellular signaling cascades, fol- (29, 30). In central neurons GABA R1 and GABA R2 are B B lowed by the induction of transcriptional regulators that rec- widely coexpressed and, a novelty for heptahelical receptors, were found to generate fully functional receptors only when * This work was supported in part by Deutsche Forschungsgemein- linked by their C-terminal tails in a heterodimeric assembly schaft Grants SFB 406, SFB 474, and SFB 269 and by the Fonds der (19 –23). Although the precise functional consequences of this Chemischen Industrie. The costs of publication of this article were association have not yet been deciphered in detail, it is thought defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted The abbreviations used are: CRE, cAMP response element; CREB, TM to the GenBank /EBI Data Bank with accession number(s) AF252627. cAMP response element-binding protein; bZIP, basic leucine zipper domain; GABA, g-aminobutyric acid; GABA § These authors contributed equally to this work. R, metabotropic B-type Supported in part by a Human Frontier Science Program postdoc- GABA receptor; Kir, inwardly rectifying potassium channel; NLS, nu- toral fellowship and by the Novartis Foundation for the Promotion of clear localization signal; SFV, Semliki forest virus; Y2H, yeast-two Science. hybrid; MEKK, mitogen-activated protein kinase/extracellular signal- ‡‡ Supported by a Heisenberg fellowship. regulated kinase kinase kinase; ATF, activating transcription factor; ¶¶ To whom correspondence should be addressed: Fax: 49-551-201- GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, 1688; E-mail: akarsch@gwdg.de. phosphate-buffered saline; EGFP, enhanced green fluorescent protein. This paper is available on line at http://www.jbc.org 35185 This is an Open Access article under the CC BY license. Interaction of GABA 35186 R and ATF-4 0.5% (w/v) Triton X-100). Proteins bound to the beads were eluted by that subunit dimerization promotes proper posttranslational SDS sample buffer and analyzed by Western blotting using anti- processing, membrane targeting, and assembly into specific CREB2/ATF-4 antibodies (1:1000; Santa Cruz Biotechnology). Affinity signaling matrices in subcellular neuronal specializations (31). purification of native GABA R1 from the solubilized P2 fraction was By means of yeast-two-hybrid (Y2H) interaction cloning, bio- performed using an aliquot of the P2 fraction. The P2 (8 mg of protein) chemical, and functional reporter assays, as well as immuno- fraction was solubilized for 1 h with 1.5% Triton X-100 in a 3-ml final cytochemistry, we now provide evidence that metabotropic volume of Tris-buffered saline (25 mM Tris, pH 7.4, 150 mM NaCl, and protease inhibitors). After ultracentrifugation at 100,000 3 g for1h, GABA Rs are also capable of directly interacting with tran- the supernatant was incubated for 5 h with 30 ml of glutathione beads scription factors and thus may utilize a mechanism for tran- preloaded with either GST or GST-ATF-4 (150 mg). Bound material was scriptional regulation unique to membrane receptors. Our mu- recovered from glutathione beads after washing four times with Tris- tational analysis indicates that GABA R bind to ATF-4, a buffered saline containing 0.2% (w/v) Triton X-100 and one wash with transcription factor of the leucine zipper ATF/CREB family, via Tris-buffered saline alone. Bound proteins were then eluted with SDS their C-terminal leucine zipper motifs, which in vivo may result sample buffer. Proteins were analyzed by Western blotting using a polyclonal goat anti-GABA R1 antibody (Santa Cruz Biotechnology). in the regulation of gene transcription upon stimulation. B Miscellaneous Methods—SDS-polyacrylamide gel electrophoresis EXPERIMENTAL PROCEDURES was performed on 6%, 10%, or 12% polyacrylamide gels. For Western Yeast Two-hybrid Screening—Two independent Y2H assays were blot analysis, proteins were transferred to nitrocellulose membranes used in parallel. Both the MATCHMAKER II (CLONTECH) and the (Schleicher & Schuell). First antibodies were overlaid with goat anti- LexA (OriGene Technologies) systems were used to screen rat brain rabbit or anti-mouse horseradish peroxidase-coupled secondary anti- cDNA libraries constructed with the activation domain vectors pAD- bodies, and chemiluminescence was detected using the Pico detection GAL4 and pJG4 –5, respectively, using amino acids 848 –960 of the kit (Pierce). C-terminal coding region of GABA R1a (18) as bait. C-terminal baits In Vivo Reporting Systems —The trans PathDetect reporting sys- were amplified by polymerase chain reaction from a rat brain library tem (Stratagene) was used to test if ATF-4 was involved in activating and inserted into the DNA-binding domain vector pGBT9 and the transcriptional expression upon receptor stimulation. For all experi- galactose-inducible vector pGilda, respectively. Screening with the ments HEK293-GBR cells were used that had been stably tranfected pGBT9 and pGilda bait yielded colonies that grew on the corresponding with GABA R1 and GABA R2 subunits. A fusion transactivator pro- B B selection plates complemented with 10 mM 3-aminotriazole for the tein between ATF-4 (or constitutively MEKK-activated Jun) and the MATCHMAKER system and were positive in the b-galactosidase assay. GAL4 DNA binding domain was constructed and transfected (pFA- Isolated plasmids were sequenced on both strands using the ABI ATF-4; 50 ng) into HEK293-GBR cells together with vectors carrying PRISM sequenase dye terminator kit on an automatic sequencer. For the luciferase reporter gene under the control of the GAL4 promoter analysis of the interaction site, GABA R1 and ATF-4 deletions were (pFR-Luc; 1 mg) and a vector expressing b-galactosidase (50 ng). 24 h generated by polymerase chain reaction with specific oligonucleotides after transfection, cells were treated with serum-free medium, and 18 h and subcloned into pGBT9 and pAD-GAL4. Yeast strains HF7c and later lysed with 500 ml of lysis buffer and the supernatant centrifuged EGY 48 were cotransformed with 100 ng each of bait and prey vector, to remove cell debris. Luciferase activity was measured with a lumi- streaked out on agar plates lacking tryptophan, leucine, and histidine nometer (Luciferase Reporter Gene Assay, Roche Diagnostics). (MATCHMAKER), and also lacking uracil (LexA). Colony growth/acti- Electrophysiology—For expression in Xenopus laevis oocytes, capped vation of the HIS3 and LEU reporter genes, respectively, as well as run-off poly(A ) cRNA transcripts were synthesized from GABA R1a, b-galactosidase activity were controlled after 4 days. GABA R2, ATF-4, and Kir3.1/3.2 concatemers (32) and ;3 ng of each Preparation of Brain Homogenates —Rat cerebral cortices were ho- injected in defolliculated oocytes. Oocytes were incubated at 19 °C in mogenized in a Teflon glass Potter homogenizer with 12 strokes at 900 ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl ,1mM CaCl ,5mM 2 2 rpm in 20 ml of ice-cold 0.32 M sucrose, 4 mM HEPES/NaOH, pH 7.3, HEPES, pH 7.4 -7.5), supplemented with 100 mg/ml gentamicin and 2.5 containing Complete and a protease inhibitor mixture (Roche Diagnos- mM sodium pyruvate, and assayed 72 h after injection. Two-electrode tics). The homogenate was centrifuged for 10 min at 800 3 g in a Sorvall voltage-clamp measurements were performed with a Turbo Tec-10 C SS-34 rotor (DuPont). The resulting pellet was used as crude nuclear amplifier (npi) and sampled through an EPC9 (Heka Electronics) inter- fraction (P1). The supernatant was recovered and spun at 27,000 3 g for face using Pulse/Pulsefit software (Heka). Oocytes were placed in a another 30 min. The resulting pellet (P2) was resuspended in 3 ml of small volume perfusion chamber and bathed with ND96 or “high K ” homogenization buffer and frozen until needed. solution (96 mM KCl, 2 mM NaCl, 1 mM MgCl ,1mM CaCl ,5mM 2 2 Bacterial Recombinant Fusion Proteins —Glutathione S-transferase HEPES, pH 7.4 –7.5). (GST) fusion proteins of GABA R (GST-GBR1 and GST-GBR2) and Neuronal Expression via Semliki Forest Viruses (SFV) and Immuno- synaptoporin tail regions (amino acids 198 –265) were constructed in cytochemistry —Recombinant SFV harboring ATF-4 were engineered pGEX-5X-1 (Amersham Pharmacia Biotech) using specific EcoRI- and and processed as described previously (33). In brief, the cDNA of the SalI-flanked oligonucleotides. Full-length ATF-4 was fused to the C N-terminally EGFP-tagged fusion protein ATF-4-EGFP was subcloned terminus of GST in pGEX-5X-1, and maltose-binding protein (MBP- into pSFV1 (Life Technologies, Inc.). After linearization with SpeI, ATF-4) in pMAL-c2 (New England Biolabs) using EcoRI and XhoI sites, cDNA was in vitro transcribed using SP6 RNA polymerase (Roche and electroporated into Epicurian Escherichia coli BL21 (Stratagene). Molecular Biochemicals). BHK21 cells were transfected by electropora- Expression of fusion protein was induced by 1 mM isopropyl-1-thio-b-D- tion (400 V, 975 microfarads) with a mixture of 10 mg of pSFV/ATF-4- galactopyranoside for 3–5 h. Cells were broken in a French press in EGFP and pSFV-helper2, respectively. After 24 h supernatant was PBS, and soluble protein fractions were recovered in the supernatant collected and stored in 450-ml aliquots at 280 °C. Prior to treatment of after centrifugation at 100,000 3 g for 1 h and kept frozen until needed. neuronal cultures, aliquots of virus were activated by 100 ml of chymo- MBP-ATF-4 Binding to GST Fusion Proteins—Glutathione beads (30 trypsin (2 mg/ml). Primary cultures of hippocampal neurons were pre- ml) were loaded by incubation for1hat4 °CinPBS with 150 mgofGST pared from 1-day-old rats as described previously (34). Hippocampal and the fusion proteins between GST and the C termini of synaptoporin, neurons were cultured in Neurobasal A medium supplemented with GBR1 and GBR2. An extract (100 ml) of bacterially expressed MBP- B27 (Life Technologies, Inc.) in 12-well plates (2 ml in each well) for 14 ATF-4 was then added to preloaded beads in binding buffer (PBS, 0.2% days. Half of the medium was removed from each well and stored at Triton X-100, and Complete) and rotated at 4 °C for 3 h. Pelleted beads 37 °C. For infection, 20 –50 ml of activated virus was added per well. were washed three times with binding buffer and once with PBS. Bound After incubation for2hat37 °C,the virus-containing medium was proteins were eluted using SDS sample buffer and separated on a 10% replaced with stored aliquots. Expression of ATF-4-EGFP was observed SDS-polyacrylamide gel. To control for MBP-ATF-4 binding, samples 12–14 h after infection. were subjected to Western blotting with rabbit anti-MBP antibodies For immunostaining, hippocampal neurons were fixed with 2% (w/v) (1:10,000; New England Biolabs). paraformaldehyde, 0.1% (w/v) Triton X-100 and blocked with 2% (v/v) Pull-down Assays—ATF-4 pull-downs from P1 brain extract were normal goat serum. Subsequently, neurons were incubated overnight achieved by mixing 100 ml of GST or GST-GBR1 bacterial extracts with with guinea pig anti-GABA R1 (1:1000; PharMingen) and rat anti- 10 mg of brain P1 Triton extract in 10 ml of 10 mM HEPES/NaOH, pH synaptophysin (1:1000; a gift of R. Jahn, Go ¨ ttingen) antibodies, respec- 7.3, containing 0.5% (w/v) Triton X-100 for2hat4 °C. After incubation, tively. After washing with PBS, cells were incubated 1 h with Cy3- 50 ml of glutathione beads were added and the mixture was incubated conjugated IgG (1:1000; Jackson Immunoresearch Laboratories). for an additional 2 h. Glutathione beads were then recovered by cen- Coverslips were washed again with PBS, mounted on slides, and ana- trifugation and washed three times vigorously with PBST (PBS with lyzed on a confocal LSM410 microscope (Zeiss). Interaction of GABA R and ATF-4 35187 FIG.1. Amino acid sequence alignment of rat and mouse ATF-4. Shown are the leucine zipper I and II motifs, the putative mitogen-activated protein kinase phosphorylation site, and the basic DNA binding domain. The rATF-4 sequence has been deposited to GenBanky under accession no. AF252627. Retina Preparation and Immunocytochemistry—Adult albino rats were anesthetized deeply with halothane and decapitated. Eyes were enucleated and opened along the ora serrata, and the posterior eyecups with the retinae attached were immersion-fixed for 15–30 min in 4% (w/v) paraformaldehyde in 0.1 M PB, pH 7.4. After dissection retinae were cryoprotected in 10% (w/v), 20% (w/v) sucrose in PB for 1 h each and in 30% (w/v) sucrose in PB overnight at 4 °C. Pieces of retinae were mounted in freezing medium (Reichert-Jung, Bensheim, Germany), sectioned vertically at 12-mm thickness on a cryostat, and collected on slides. For double-labeling experiments, guinea pig anti-GABA R1 (1: 1000; PharMingen) and rabbit anti-CREB2/ATF-4 antibodies (1:1000; Santa Cruz Biotechnology) were used and visualized by red and green fluorescence secondary antibodies, goat anti-rabbit IgG, and goat anti- guinea-pig IgG (Alexay 594, Alexay 488; 1:500; Molecular Probes). Sections were examined by confocal laser-scanning microscopy (Leica DM IRBE; Leica Microsystems, Heidelberg, Germany) using a 363 objective and special filter settings (Leica TCS SP). FIG.2. GAL-4-based Y2H analysis of the interaction between RESULTS GABA R1 and ATF-4. A, deletion constructs of GABA R1 (amino acid B B Y2H Assay—Using the Y2H system (35), we sought to isolate region on the left) were used as baits for binding ATF-4 as prey. The HIS3 marker was used as reporter in the Y2H assay. B, the equivalent candidate proteins that directly interact with and modulate the analysis is shown for deletion constructs of rATF-4 used as prey for signaling function of GABA receptors. Therefore, the complete binding the C terminus of GABA R1. Functional sites depicted in Fig. C-terminal intracellular region of GABA R1a was initially 1are boxed in this schematic representation of ATF-4. C, helical used as bait to screen rat brain cDNA libraries. Two independ- wheel diagrams of the putative leucine zipper-based interface between GABA R1 and rATF-4. The view is from the N termini starting from ent screenings of ;2 3 10 recombinants resulted in the isola- S887 (position a in GABA R1a) and Q299 (a9 in rATF-4). Heptad tion of 27 and 180, respectively, positive clones, the open read- B positions are labeled a– g (a9–g9). ing frames of which all encoded regions of the same polypeptide. Data base analysis indicated very high similarity to the mouse transcription factor mATF-4 (36), also known as were constructed and tested for complementation in the Y2H C/ATF (37) or mTR67 (38). The complete open reading frame of assay. These experiments showed that deletion constructs in the rat orthologue (rATF-4) is shown in Fig. 1. rATF-4 is 347 the GABA R1 bait, removing partial sequences from the C amino acids in length and shares 94% and 86% amino acid terminus, allow binding of rATF-4 (Fig. 2A) until a leucine at identity with mouse ATF-4 and the human ATF-4 (hCREB2/ amino acid position 915 (Leu-915) is removed (DQ914) or ex- TAXREB67; Refs. 39 and 40), respectively. At the C terminus, changed by a glycine (L915G) or serine (L915S) residue (data rATF-4 harbors a conserved basic leucine zipper (bZIP) dimer- not shown in the illustration). In contrast, replacement of Gln- ization motif that binds CRE and is present in all CREB/ATF 916 by an alanine (Q916A) did not disturb the interaction. proteins. In addition, rATF-4 contains a second heptad repeat Further restriction analysis on the 59 end of GABA R1a as- of leucines between amino acids 89 and 124 near the N termi- signs the region of interaction to amino acids 887–915. Inter- nus, and a potential phosphorylation site for mitogen-activated estingly, this domain has been recently mapped to likely par- protein kinase (amino acid position 164). ticipate in the obligate assembly of GABA R1 and GABA R2 B B We performed control experiments (i) with bait or prey vec- subunits into heteromeric receptor complexes (23, 41), suggest- tors missing, (ii) using empty vectors, (iii) using vectors that ing a bifunctional role of this site. expressed unrelated proteins such as pRHFM1 encoding the Conversely, deletion constructs in rATF-4 demonstrated that Drosophila bicoid protein homeodomain, and (iv) using vectors the first leucine zipper an the putative mitogen-activated pro- that expressed other proteins harboring a bZIP domain, e.g. tein kinase site of rATF-4 were dispensable for binding, CREB. All these controls were negative, excluding autoactiva- whereas the C-terminal leucine zipper (amino acids 301–337) tion and corroborating the specificity of the interaction between was required for association with GABA R1 (Fig. 2B). When GABA R1 and rATF-4. For a detailed mapping of the interac- described in terms of the heptad patterns seen in a helical tion domains, deletion mutants of both GABA R1 and ATF-4 wheel diagram, the interaction sites between the C termini of B Interaction of GABA 35188 R and ATF-4 FIG.4. Expression in Xenopus oocytes and in vivo luciferase reporter assay. A, expression of GABA R1, GABA R2, ATF-4, and B B FIG.3. Interaction of GABA Rs and ATF-4 in vitro. A, bacterially Kir3.1/3.2 concatemeric channels in Xenopus oocytes elicits basal and expressed GST and GST fusion proteins (GST-Por, fusion with the agonist-dependent (as indicated by black bars)K currents in the C-terminal tail of the synaptic vesicle protein synaptoporin; GST-GBR1 absence (top left trace) and presence of injected ATF-4 antibodies (bot- and GST-GBR2, fusion proteins of the C-terminal tails of GABA R1a tom left trace). High K (96 mM, arrowheads) and 10 mM baclofen (black and R2) were immobilized on glutathione-Sepharose and then incu- bars) were applied to the bath and oocytes voltage-clamped at -80 mV. bated with recombinant MBP-ATF-4. B, GST-GBR1 or GST-ATF-4 Dashed lines indicate zero-current levels, scale bar represents 2 mA and were used to test the binding of ATF-4 (left panel) and native GBR1 10 s. The right panel shows a bar graph summary of Kir3.1/3.2 inward (right panel) present in a Triton X-100 extract from rat brain. Bound currents at -80 mV in the presence (black bars) and absence (white bars) material was eluted using SDS sample buffer, separated by SDS-PAGE, of overexpressed ATF-4 and injected anti-ATF-4 antibodies. B, in vivo and immunoblotted with anti-MBP (A), anti-ATF-4 (B, left), or anti- luciferase trans-reporter assay performed on HEK293 cells stably GBR1 (B, right). transformed with GABA R1 and GABA R2. Fusion transactivator pro- B B teins between the GAL4 DNA binding domain and ATF-4 and Jun (activated with MEKK), respectively, were transfected together with ATF-4 and GABA R1 conform with good approximation to the luciferase reporter gene under the control of the GAL4 promoter. classic coiled-coil structures (Fig. 2C). In the bZIP domain of Luciferase activity was measured in the presence or absence of 10 mM baclofen. rATF-4, the periodic array of leucines at every seventh position likely interdigitates with that of a matching helix formed by the GABA of rATF-4, was detected exclusively in the GST-GBR1, but not R1 C terminus to form a zipper-like structure. It has been suggested earlier that bZIP domains not only mediate in the GST eluates (Fig. 3B, left). In parallel, we showed that association between transcription factors prior to DNA binding ATF-4 can bind to native GABA R1 by performing similar (42), but also form coiled-coil structures from up to four helices pull-down experiments using immobilized GST or GST-ATF-4 with various other proteins (43). Together with the array of and Triton X-100 extract from crude P2 rat brain fraction. leucines at position d in GABA R1 and d9 in ATF-4, several Bound material was eluted and analyzed by Western blotting using anti-GABA R1 antibodies. A 130-kDa protein was de- features are consistent with such an interaction: (i) the b-branched amino acids valine, isoleucine (and alanine) occur tected exclusively in the GST-ATF-4, but not in the GST elu- at the alternate hydrophobic positions a and a9; (ii) there are ates (Fig. 3C). highly conserved breaks at these positions caused by polar Electrophysiology and in Vivo Reporting System—In a fur- asparagines; and (iii) the amino acids preceding the alternate ther series of experiments with heterologously expressed pro- juxtaposed hydrophobic residue and following the leucine of the teins, we sought to reveal a functional consequence of the next heptad are often oppositely charged in both proteins to interaction between GABA R and ATF-4. First, we investi- allow electrostatic interactions (38). Consistent with our pull- gated whether cytosolic ATF-4, when binding to the dimeric down experiments (see below), helical wheel representation of receptor, might provide a negative regulator of receptor func- GABA R2, but not the C-terminal splice variant GABA R1d tion in that it interferes with G protein binding and thus classic B B short term signaling. This putative role of ATF-4 on G protein (24), which misses the leucine zipper, does also reveal comple- mentarity to ATF-4 as required for stable dimerization. signaling of the receptors was studied in Xenopus oocytes under Biochemical Assays—To verify the interaction between two-electrode voltage-clamp. Coinjection at a 1:1:1 ratio of ATF-4 and GABA R1a by an independent assay, we evaluated cRNAs for GABA R1, GABA R2, and Kir3.1/3.2 concatemers B B B as target channels for receptor signaling resulted in the expres- binding of the respective bacterially expressed fusion proteins. In addition, we have tested the binding of C-terminal regions of sion of large basal and baclofen-induced inwardly rectifying K GABA currents as described previously (17). Two days after injection, R2 and the unrelated synaptic protein synaptoporin (Por). GST and the GST-fusion proteins GST-GBR1, GST- Kir3 current amplitudes activated by baclofen (10 mM) aver- GBR2, and GST-Por were immobilized on glutathione beads aged 26.0 6 4.5 mA(n 5 7) at 280 mV in the presence of 96 mM and incubated with either MBP or MBP-ATF-4 bacterial ly- external K (Fig. 4A). To test for the role of endogenous ATF-4, sates. After washing the resin, bound material was eluted and anti-ATF-4 antibodies were injected 1 h prior to the experiment analyzed by Western blotting using anti-MBP antibody. MBP- at a dilution of 1:100. In the presence of antibody, the activa- ATF-4, but not MBP (data not shown), specifically interacted tion kinetics of baclofen-induced Kir currents (as determined with both immobilized GST-GBR1 and GST-GBR2, but not from responses to hyperpolarizing voltage steps) was un- with GST or GST-Por (Fig. 3A). changed, but mean amplitudes were slightly increased by sta- For demonstrating that GABA R1 is capable to interact with tistically significant 18% (p , 0.05). Similarly, when ATF-4 was overexpressed under otherwise unchanged recording con- rATF-4 present in rat brain, we performed pull-down experi- ments with immobilized GST or GST-GBR1 using crude P1 rat ditions, ligand-activated currents in the presence of antibody brain extracts. Triton X-100 extracts from a crude rat brain (25.8 6 2.4 mA) were elevated by 12% compared with control nuclear fraction was incubated with GST or GST-GBR1, and conditions (25.1 6 2.1 mA, n 5 7), indicating that the presence protein binding to the fusion proteins was recovered by adding of intrinsic or exogenously expressed ATF-4 plays only a mar- glutathione beads. Bound material was eluted and analyzed by ginal role in receptor-stimulated G protein activation. Western blotting using anti-rATF-4 antibodies. A doublet of Next, we employed the PathDetect luciferase reporting sys- 95–100-kDa proteins, corresponding most probably to a dimer tem to investigate the in vivo consequences of the GABA R/ B Interaction of GABA R and ATF-4 35189 FIG.5. Colocalization of ATF-4 and GABA R1a in hippocampal neurons. Confocal fluorescence micrographs of primary hippocampal cells immunostained with anti-GABA R1 antibodies and detected with Cy3-labeled secondary antibody (A). B, EGFP-tagged ATF-4 expressed through a recombinant Semliki forest virus. C, fluorescence overlay in the detail figure of regions boxed in A and B shows complete cocluster- ing of ATF-4 and GABA R1 in the dendritic membrane. D, fluorescence overlay of ATF-4-EGFP (green) and antibody-labeled synaptophysin (Syn, Cy3 signal in red). Scale bars,20 mm(A and B), 10 mm(C), and 7 mm(D). ATF-4 interaction on transcriptional activation in HEK293 cells that were engineered to stably express GABA R1 and GABA R2. In the trans-reporting assay, the GAL4 DNA bind- ing domain was joined to ATF-4 and the construct cotrans- fected with a GAL4-driven luciferase reporter plasmid. Cells were assayed after 18 h in serum-free medium. Although re- sults varied considerably between different wells, we found that stimulation with 50 mM baclofen for 1 h increased lucifer- ase levels to 144 6 72% (n 5 10) compared with unstimulated cells (Fig. 4B), which is a statistically significant increase (Stu- dent’s t test; p , 0.05). In another experiment, the GAL4 DNA binding domain was fused to c-Jun and stimulated with con- stitutively activate MEKK. Under these conditions stimulation with baclofen significantly decreased luciferase levels to 56 6 16% (Fig. 4B). The precise mechanisms underlying this differ- ential receptor signaling are presently not understood, but are in accordance with previous findings, if we assume that endog- enous ATF-4 is also present in HEK293 cells. ATF-4 has been FIG.6. Colocalization of GABA R1 and ATF-4 in an amacrine found to suppress the transcriptional action of Jun family cell of the rat retina. Confocal laser-scanning micrographs of a trans- members (44), but is a transcriptional activator by itself (Ref. verse section of rat retina showing an amacrine cell in the inner nuclear 45; see “Discussion”). layer (INL) coimmunostained for GABA R1 (green) and ATF-4 (red). In Neuronal Expression via SFV—The cellular expression of the merged panel below, coexpression of the two proteins is clearly visible in the cell’s cytoplasm and along its processes in the inner GABA R1 and ATF-4 in vivo was first analyzed in low density plexiform layer (IPL; yellow-orange). Scale bar,10 mm. rat hippocampal cultures by immunofluorescence. Primary cul- tures of rat hippocampal neurons were infected with an SFV vector containing EGFP-tagged ATF-4 (ATF-4-EGFP) and ex- prominent baclofen-induced inwardly rectifying K currents pression evaluated 12–18 h after infection. In confocal images (data not shown). After staining with Cy3-labeled secondary ATF-4 was found in the nucleus and diffusely in the cytoplasm, antibodies, GABA R1 antibodies gave rise to a punctate mem- but primarily clustered at specific sites in the outer membranes brane pattern of dendritic immunoreactivity that exactly colo- of hippocampal cell somata and along dendrites (Fig. 5B). Un- calized with coexpressed ATF-4 (Fig. 5, A and C). A similar der whole cell patch-clamp conditions, all hippocampal neurons pattern of GABA R immunoreactivity was obtained in hip- cultured from this age displayed GABA Rs as revealed by pocampal cells that were not infected with ATF-4 SFV, ensur- B Interaction of GABA 35190 R and ATF-4 ing that overexpression of ATF-4 did not affect receptor only after agonist-driven release. So far, the functional assays distribution. performed in our study do raise several unsolved questions on In many cases these puncta were not congruent with synap- the stimuli and targets of cytosolic ATF-4. Although trans- tic regions, as indicated by the differential distribution of the fected COS cells and primary neurons in our experiments were presynaptic terminal marker synaptophysin (Fig. 5D) or glu- depleted overnight of serum and other extracellular stimuli, tamic acid decarboxylase that shows inhibitory GABAergic ter- prominent ATF-4 signals often remained in the nuclei. This minals (data not shown). Our findings suggest that GABA R1 may have masked a pronounced, visually detectable transloca- and ATF-4 are clustered predominantly at extrasynaptic sites tion signal of ATF-4 upon receptor stimulation with baclofen. in neuronal cells. As an alternative explanation, ligand binding to the receptor Colocalization in the Retina—For the mammalian retina, it may be insufficient to fully activate ATF-4 as a transcriptional has been shown that GABA Rs are strongly expressed in am- protein, but require a coincident stimulus through another acrine cells both pre- and postsynaptically (46). We therefore signaling protein of the receptor matrix. investigated whether GABA Rs and ATF-4 would indeed co- When using the trans luciferase reporter assay, a moderate cluster at distinct subcellular sites in a native central nervous rise in transcriptional activity via ATF-4 was seen upon recep- system neuron. Fig. 6 depicts a wide field amacrine cell with tor stimulation. This trend was supported by a cis reporting the cell body located in the inner nuclear layer and its processes assay in HEK-GBR1 stable cells, in which CRE-driven lucifer- stratifying in the inner plexiform layer close to the inner nu- ase expression was enhanced by ;20% by baclofen in the pres- clear layer. Immunocytochemical double-labeling experiments ence of 10 ng/ml pertussis toxin (data not shown). In this assay with anti-GABA R and anti-ATF-4 antibodies revealed the B gene expression was reduced by .30% in the absence of per- striking overlap of GABA R immunoreactivity (green) and tussis toxin which disrupts receptor stimulation of G proteins. ATF-4 immunoreactivity (red) both in the cytoplasmic compart- This indicates that, even under experimental suppression of G ment of the amacrine cell and also along its processes and protein-mediated GABA R signaling, ATF-4 may be involved terminal arborizations, suggesting a strong functional relation- in CRE-mediated stimulation of gene expression independent ship between these two proteins in selected neurons. of G proteins. Yet, this action is far from being understood in detail. Similar to other proteins of the CREB/ATF family, ATF- DISCUSSION 4/CREB2 is known to bind to the transcriptional enhancer Here we demonstrate for the first time a tight and direct motif CRE as homo- or heterodimers in conjunction with c-Jun, interaction between a heptahelical neurotransmitter receptor but also together with TATA-binding protein, TFIIB, RAP30 and a soluble transcription factor (ATF-4). We show that the subunit of TFIIF, or the coactivator CREB-binding protein bZIP domain of ATF-4 associates with the GABA R C termini CBP/p300 (38, 45, 52). It has been reported that ATF-4, like in a coiled coil-confirmation, which is common, e.g., among CREB, can act as a transcriptional activator (45), but under a structural proteins and contains between two and four helices. variety of experimental conditions significantly represses CRE- Given that the site at which ATF-4 interacts with both GAB- dependent transcription (39, 44). This bifunctional role may be R1 and likely GABA R2 overlaps with the putative interac- B B explained by the displacement of other CREB activator/coacti- tion site between the two subunits (23, 41), it may be hypoth- vator proteins from the CRE promoter site by high amounts of esized that, at least temporarily, triple-helix structures exist. It ATF-4 (squelching), resulting in inhibition of transcriptional is currently thought that individually neither of the two recep- activation. A repressive action of ATF-4 orthologues on tor subtypes is expressed and transported with high efficiency CREB1-mediated transcription in Aplysia, Drosophila, and ro- to the outer membrane, because of homodimer instability (41). dents (53) has been interpreted to impede synaptic plasticity Instead, the majority of native GABA Rs are likely to exist as and affect spatial and social learning (54, 55). Based on its heterodimers between GABA R1 and GABA R2 (19 –23) with B B repressive character in the nervous system and in analogy to specific electrostatic interactions in their C termini giving rise the function of tumor suppressor genes, CREB2/ATF-4 has to parallel coiled-coil a-helices. Our assays convincingly been suggested to act as a “memory suppressor gene” that showed that ATF-4 is tightly linked to GABA Rs expressed in decreases synaptic strength or removes inhibitory constraints neuronal membranes. Similarly, overlapping edge fluorescence of long term memory storage (56, 57). of ATF-4 and GABA R1 immunoreactivity was observed by Heptahelical receptors are commonly thought to signal pri- confocal microscopy after cotransfection into COS-7 cells (data marily through coupling to G proteins. Only recently, however, not shown). In COS-7 cells that lack GABA R1 and GABA R2, B B they have been found to interact with a growing number of ATF-4 was not seen at the plasma membrane, but only diffuse membrane and cytosolic proteins, including proteins that func- cytoplasmic staining was found, suggesting that GABA Rs tion in signal termination (58), synaptic targeting (59), mito- play a role in recruiting ATF-4 to the outer plasma membrane. genic signaling (60, 61), and translational regulation (62, 63). The notion that in neurons transcription factors are localized in With the direct interaction of metabotropic GABA R and the dendrites and may be retrogradely transported to the nucleus ubiquitously expressed transcription factor ATF-4 (39) demon- has emerged only recently (47). One of the possible cellular strated here, a novel alternative mechanism by which tran- consequences of the documented GABA R/ATF-4 interaction scriptional regulation important for long term memory forma- would be that agonist stimulation of the receptor at the outer tion (64) may be initiated at inhibitory synapses of the surface membrane releases ATF-4, which then translocates mammalian central nervous system, is emerging. into the nucleus to increase the nuclear pool of this transacti- vating protein. Like other transcription factors, ATF-4 is likely Acknowledgments—We are grateful to Drs. B. Bettler and F. Do ¨ ring imported across the nuclear membrane by shuttling proteins for continuous intellectual and experimental support as well as D. that recognize nuclear localization signals (NLS; Refs. 48 and Reuter, A. Niehuis, D. Magalei, A. Hildebrand, and O. Dick for excellent technical assistance. We also thank I. Herford and Dr. C. Rosenmund 49). 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Published: Nov 1, 2000

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