The GABAB receptor interacts directly with the related transcription factors CREB2 and ATFx

Abstract

gamma-Aminobutyric acid type B (GABA(B)) receptors mediate the metabotropic actions of the inhibitory neurotransmitter GABA. These seven-transmembrane receptors are known to signal primarily through activation of G proteins to modulate the action of ion channels or second messengers. The functional GABA(B) receptor is made up of a heterodimer consisting of two subunits, GABA(B)-R1 and GABA(B)-R2, which interact via coiled-coil domains in their C-terminal tails. By using a yeast two-hybrid approach, we have identified direct interactions between the C-terminal tails of GABA(B)-R1 and GABA(B)-R2 with two related transcription factors, CREB2 (ATF4) and ATFx. In primary neuronal cultures as well in recombinant Chinese hamster ovary cells expressing GABA(B) receptors, CREB2 is localized within the cytoplasm as well as the nucleus. Activation of the GABA(B) receptor by the specific agonist baclofen leads to a marked translocation and accumulation of CREB2 from the cytoplasm into the nucleus. We demonstrate that receptor stimulation results in activation of transcription from a CREB2 responsive reporter gene. Such a signaling mechanism is unique among Family C G protein-coupled receptors and, in the case of the GABA(B) receptor and CREB2, may play a role in long-term changes in the nervous system.

Figure 1

Interactions between CREB2, ATFx, and GABA_B receptor subunits, as determined by β-galactosidase activity in the YTH system. CREB2 and ATFx GAL4_AD fusion protein and GABA_B receptor C termini GAL4_BD fusion proteins were coexpressed in the YTH system, and β-galactosidase activity was determined. The regions of the GABA_B-R1 expressed were amino acids 854–961 (GABA_B-R1a) and amino acids 877–928 for the coiled-coil (CC) domain. The amino acids expressed from the GABA_B-R2 C terminus were 744–941 and 779–847 for the coiled-coil domain. The intensity of the resulting β-galactosidase activity is indicated by the number of + signs. The relative positions of the bZIP domain and the putative MAPK phosphorylation sites are shown as hatched boxes and star symbols, respectively.

Figure 2

Interaction of GABA_B-R1 and GABA_B-R2 with the Transcription factor CREB2. (a) Myc-GABA_B-R1b and Flag-CREB2 coimmunoprecipitation (I.P.) from HEK293T cells, transfected with Myc-GABA_B-R1b and Flag-CREB2. GABA_B-R1 and CREB2 were immunoprecipitated by 9E10 (Myc) antisera (lanes 1 and 2) or anti-Flag antisera (lanes 3 and 4), and the precipitates were immunoblotted with anti-Flag antisera. Cotransfection of cells with GABA_B-R2 (lane 1 and 3) blocked coimmunoprecipitation of GABA_B-R1 and CREB2. GABA_B-R2 coexpression had no effect on the direct immunoprecipitation of FLAG-CREB2 with anti-Flag antisera (lanes 3 and 4). (b) GABA_B-R1 and CREB2 coimmunoprecipitation from rat-brain homogenates. Immunoblots of total lysate (Lys), together with immunoprecipitates against affinity purified GABA_B-R1 antisera (R1) or pooled Sheep normal IgG (Con), are shown. Lysates show the presence of GABA_B-R1 and CREB2, whereas the GABA_B-R1 antisera coprecipitated a strong CREB2 immunoreactive band at the expected size. (c) Coexpression of CREB2 with GABA_B-R1 and GABA_B-R2 receptors in HEK293T cells does not affect GABA-mediated stimulation of [³⁵S]GTPγS-binding activity. GABA stimulation of [³⁵S]GTPγS binding in cells transfected with GABA_B-R1b and GABA_B-R2 in the absence (□) and presence of either CREB2 (●) or eGFP-CREB2 (▴) or in combination with GABA_B-R1b together with CREB2 (▵) or eGFP-CREB2 (○). The data shown are the means ± SD of triplicate measurements and are representative of three independent experiments.

Figure 3

Codistribution of GABA_B-R1 and CREB2 in neurons. (a) Codistribution of GABA_B-R1 and CREB2 in rat primary cortical cultures. Fixed rat-cortical neurons (E17) were stained with primary antibodies for GABA_B-R1 and CREB2. Images were viewed for GABA_B-R1 (green) and CREB2 (red), and the images were merged. [bar = 10 μm]. Arrowheads indicate areas of distinct codistribution for the two proteins in the distal dendrites. (b) Codistribution of GABA_B-R1 and CREB2 in rat-cerebellar cortex. Fluorescence was detected by confocal microscopy for GABA_BR1 (green) and CREB2 (red), and the images were merged. Arrows indicate Purkinje cells, and the arrowhead shows granule cells. [bar = 15 μm]. (c) Distinct non-codistribution of GABA_B-R1 and GFAP in rat-cerebellar cortex. Fluorescence was detected by confocal microscopy for GABA_BR1 (green) and GFAP (red), and the images were merged. [bar = 15 μm].

Figure 4

GABA_B receptor activation alters CREB2 localization in CHO-GABA_B cells. (a) CHO-ORL1 cells show bright nuclear localization of eGFP-CREB2. (b) CHO-GABA_B cells show diffuse and punctate cytoplasmic fluorescence for eGFP-CREB2 as well as bright nuclear fluorescence. Treatment with 100 μM baclofen significantly decreases cytoplasmic eGFP-CREB2. (c) GABA_B antagonist CGP54626 (1 μM) blocks the effects of baclofen (100 μM). (d) Immunoblots of cytoplasmic extracts prepared from CHO-GABA_B cells, transfected with CREB2, show decreased levels of CREB2 in the cytoplasm after baclofen treatment (100 μM), whereas CGP54626 (1 μM) partially blocked the decrease in CREB2 levels. Cytoplasmic levels of GABA_B-R1 remained constant.

Figure 5

Baclofen increases CREB2 levels in the nuclei in primary rat cortical cultures. Treatment of cortical cultures with baclofen (20 μM) gave a 3-fold increase in CREB2 immunoreactivity in nuclear extracts in contrast to cytoplasmic extracts, as determined by Western blotting. The nuclear localization of CREB2 was blocked by the GABA_B receptor antagonist CGP54626 (1 μM). Sc-35 localized entirely to the nuclear extract and showed no alteration in levels with baclofen treatment. Means of four independent experiments reveal a 3.3 ± 1.3 (SD) increase in nuclear CREB-IR after baclofen treatment.

Figure 6

CREB2 activates Gadd153 transcription through the GABA_B receptor. (a) Dose-response to baclofen. Transfected CHO-GABA_B cells were treated with baclofen (0.1 μM-1 mM; 6 h) and cytoplasmic extracts were prepared. CAT levels, from the Gadd153-CAT reporter gene (14), were determined by ELISA assay. Baclofen induced CAT expression in a dose-dependent manner with an EC₅₀ of 4 μM. (b) Effects of CGP54626, PTX, and PD98059 on baclofen-induced CAT production. Cells were pretreated with CGP54626 (1 μM; 30 min), PTX (50 ng/ml; 16 h), or PD98059 (50 μM; 30 min) and CAT levels were determined in cytoplasmic extracts after baclofen treatment (100 μM; 6 h). All three compounds blocked baclofen/CREB2-mediated CAT expression. Transfection of cells with vector (pcDNA3) instead of CREB2 greatly diminished CAT expression levels, and resulted in a markedly decreased response to baclofen.