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Originally published In Press as doi:10.1074/jbc.M603118200 on June 5, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22180-22189, August 4, 2006
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Modulation of GABAA Receptor Phosphorylation and Membrane Trafficking by Phospholipase C-related Inactive Protein/Protein Phosphatase 1 and 2A Signaling Complex Underlying Brain-derived Neurotrophic Factor-dependent Regulation of GABAergic Inhibition*

Takashi Kanematsu{ddagger}, Atsushi Yasunaga{ddagger}, Yoshito Mizoguchi{ddagger}§1, Akiko Kuratani{ddagger}2, Josef T. Kittler, Jasmina N. Jovanovic||, Kei Takenaka**, Keiichi I. Nakayama{ddagger}{ddagger}, Kiyoko Fukami§§, Tadaomi Takenawa**, Stephen J. Moss¶¶, Junichi Nabekura§, and Masato Hirata{ddagger}3

From the {ddagger}Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan, the §Department of Developmental Physiology, National Institute for Physiological Science, Okazaki 444-8585, Japan, the Department of Physiology, University College London, London WC1E 6BT, United Kingdom, the ||Department of Pharmacology, School of Pharmacy, Brunswick Square, London, WC1N 1AX, United Kingdom, the **Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, the {ddagger}{ddagger}Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan, the §§Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan, and the ¶¶Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, April 3, 2006 , and in revised form, June 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The neurotrophin brain-derived neurotrophic factor (BDNF)4 has been shown to modulate directly both excitatory and inhibitory synaptic transmission. Acting via the TrkB tyrosine kinase receptor, BDNF exerts rapid effects both presynaptically, by modulating transmitter release, and postsynaptically, by changing the properties of ionotropic receptors (1). At inhibitory synapses, acute application of BDNF depresses inhibitory synaptic transmission mediated through {gamma}-aminobutyric acid type A (GABAA) receptors in hippocampal slices (2) and reduces miniature inhibitory postsynaptic currents acutely or following a transient increase, in cultured hippocampal (3, 4) and cerebellar granule neurons (5). Modulation of the strength of synaptic inhibition by BDNF may be important for the maturation of inhibitory synapses (6) and, in addition, may have important implications for synaptic plasticity and information processing in the brain. Two possible mechanisms for the BDNF-dependent regulation of GABAA receptor function have been proposed: alterations in GABAA receptor cell surface numbers (3, 5) and/or modulation of GABAA receptor phosphorylation (4), which may be mutually related (7). However, the precise mechanism regarding the molecular machinery underlying these events requires further investigation.

Phospholipase C-related but catalytically inactive protein type 1 (PRIP-1), a novel D-myo-inositol 1,4,5-trisphosphate-binding protein, is a molecule similar to phospholipase C-{delta}1 but is catalytically inactive and is expressed predominantly in the brain (8-15). PRIP-1 has a number of binding partners, including the catalytic subunit of protein phosphatase 1{alpha} (PP1c) (16, 17), GABAA receptor-associated protein (18, 19), and GABAA receptor beta subunits (17). We have recently reported that PRIP-1 plays an important role in regulating GABAA receptor activity based on pharmacological and behavioral phenotype of mice lacking the PRIP-1 gene (PRIP-1 KO) (19). In addition, PRIP-1 activity is important for phospho-dependent modulation of GABAA receptors in response to cAMP-dependent protein kinase A signaling pathways by modulating the binding and phosphatase activity of PP1 (17). In addition to PRIP-1, the identification of the second PRIP isoform, PRIP-2, with a broad tissue distribution, including the brain, has been reported recently (20-22). PRIP-2 also interacts with both PP1c and GABAA receptor-associated protein (22, 23), but the contribution of PRIP-2 to signaling pathways regulating GABAA receptors is currently unknown (24). Given the functional similarity between PRIP-1 and -2 proteins at least in vitro, it is of interest to investigate the regulation of GABAA receptors in the absence of both isoforms. PRIP-1 and -2 double knock-out (PRIP-DKO) mice were therefore generated to help establish the functional significance of these proteins in GABAA receptor phosphorylation and trafficking.

In the present study, we examined the specific role(s) of PRIP proteins in BDNF-mediated modulation of GABAA receptors. We observed a complete lack of BDNF-dependent inhibition of GABA-evoked currents in PRIP-DKO mice because of altered properties of the BDNF signaling pathway regulating phosphorylation and cell surface expression of GABAA receptors. Collectively, our results suggest that PRIP-1 and -2 play important roles in the modulation of GABAA receptor function and may be involved in long term changes in the efficacy of inhibitory synaptic transmission.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of PRIP-DKO Mice—The PRIP-1 KO mice (19) and PRIP-2 KO mice (24) that were both back-crossed against the C57BL/6J background (n = 7 and n = 2, respectively), were crossed to generate a PRIP-DKO mouse strain and corresponding wild type (WT). Homozygous PRIP-DKO and WT mice were mated inter se to obtain the required number of mice, and only F1 and F2 generations of both genotypes were used for experiments. The handling of mice and all procedures performed on them were approved by the Animal Care Committee of Kyushu University, following the guidelines of the Japanese Council on Animal Care.

Neuron Culture Preparation—High density cultures of dissociated hippocampal or cortical neurons were prepared from both WT and PRIP-DKO mice (postnatal day (P)0 or P1) as described previously (19). Neurons were cultured for 14-21 days (14-21 days in vitro (DIV)) before experiments.

Electrophysiological Recordings—Cultured hippocampal neurons (14-18 DIV) were used for electrophysiological recording. Electrical measurements were performed by the nystatin-perforated patch recording method described in Mizoguchi et al. (25). The synthetic peptide incorporating residues 553-566 of rat PRIP-1 (EGEVTDEDEEAEMS in an abbreviation by a single character of amino acids) or scrambled control peptide (EETEMDAGDEVSEE) were added to the pipette solution at a concentration of 3 µg/ml for the measurement of GABA-evoked Cl- currents (IGABA). All of the experiments were performed at the room temperature (25 ± 1 °C). All of the data are normalized to the amplitude just before BDNF application and expressed as the means ± S.E.

Generation of Plasmids—A series of C-terminal truncated rat (r)PRIP-1 constructs (see Fig. 5A) was constructed in pSG5 vector. A series of N-terminally truncated rPRIP-1 and N-terminally truncated human (h)PRIP-2 constructs (see Fig. 5A) were generated in pETHis6-30 vector (19). To make rPRIP-1/pDsRedN1, a pcMT31 clone (9) was introduced into a pDsRedN1 vector (Clontech Laboratories, Palo Alto, CA). For construction of a PRIP-1-binding peptide plasmid that disrupts the association between PRIP and beta subunits, the PCR-amplified fragment (amino acid residues 553-565 of rPRIP-1) was introduced into pIRES2-EGFP vector (Clontech Laboratories). Constructing strategies of Myc-tagged PP2A in pRK5 vector, GABAA receptor {alpha}1Myc and beta2Myc subunit in pGW1 vector, and glutathione S-transferase (GST)-fused intracellular loop regions of {alpha}1, beta1, beta2, and beta3 subunits were previously described (4, 26, 27).

Immunoblot Analysis of GABAA Receptor beta3 Subunits Phosphorylation—To assess changes in phosphorylation of GABAA receptors, anti-phospho(P)-beta3 antibody, which recognizes the phosphorylated Ser408/Ser409 residues in the beta3 subunit (4), was used for immunoblot analysis. The procedures for this analysis used were similar to those described previously (4). Primary antibodies used were as follows: rabbit anti-PRIP-1 antibody (19), rabbit anti-PRIP-2 antibody (24), rabbit anti-PP1 antibody, mouse anti-beta2/3 antibody (clone 62-3G1), rabbit anti-pan PKC antibody that recognizes PKC {alpha}, beta, and {gamma} (number 06-870; Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY), mouse anti-TrkB antibody (clone 47), mouse anti-PP1 antibody (clone 24), mouse anti-PP2A antibody (clone 46) (BD Transduction Laboratories, Lexington, KY), mouse anti-beta tubulin antibody (Roche Applied Science), and mouse anti-Myc antibody (9E10). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Amersham Biosciences) were used as secondary antibodies. Prestained protein markers (broad range; New England BioLabs, Beverly, MA; precision plus, Bio-Rad) were used. The chemiluminescent signals were detected and quantified using a LAS-1000 plus gel documentation system (Fujifilm, Tokyo, Japan).

Immunoprecipitation and GST Protein Pulldown Assay—Co-immunoprecipitation assays and GST pulldown assays were performed as described previously (17, 19). Briefly, the lysate obtained from COS7 cells transfected with the full-length rPRIP-1/pSG5 and PP2Ac/pRK5-Myc plasmid, cultured rat cortical neurons (21 DIV), or mouse brains was subjected to immunoprecipitation with rabbit control IgG, rabbit polyclonal anti-Myc antibody (c-Myc (A-14); Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit polyclonal anti-PRIP-1 antibody, followed by the addition of 20 µl of 50% slurry of protein G-Sepharose beads (Amersham Biosciences). For GST protein pulldown analysis, GST-{alpha}1, -beta1, -beta2, and -beta3 and GST immobilized on glutathione-Sepharose beads were incubated with recombinant proteins of interest or with the rat brain lysate. A series of recombinant PRIP-1 and PRIP-2 mutants (see Fig. 5A) were expressed and labeled by in vitro transcription/translation using the TranscendTM chemiluminescent translation detection system and analyzed by Western blotting with streptavidin-horseradish peroxidase (Promega, Madison, WI).

Immunocytochemistry—For detection of GABAA receptors expressed at the cell surface, COS7 or HEK293 cells were transfected with rPRIP-1/pDsRedN1, GABAA receptor {alpha}1Myc and beta2Myc subunit constructs in pGW1 and PRIP-1-binding peptide in pIRES2-EGFP, using the procedure described previously (28). Co-localization between PRIP-1 and GABAA receptors in cultured neurons was investigated as follows: mouse anti-GABAA receptor beta2/3 antibody (clone 62-3G1) or mouse anti-beta subunits antibody (MAB341; Chemicon Inc., Pittsburgh, PA) was added to the culture medium to bind to cell surface-expressed beta subunits prior to fixation with 4% paraformaldehyde and subsequent permeabilization using 0.1% saponin in 80 mM PIPES, pH 7.2, 1 mM MgCl2, 1 mM EGTA. The cells were then stained with rabbit anti-PRIP-1 antibody followed by incubation with Cy3-conjugated anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa-488-conjugated anti-rabbit antibody (Molecular Probes, Eugene, OR). The signals were visualized by confocal microscopy (Bio-Rad).

Cell Surface Receptor Assay ([3H]Muscimol Binding Assay)—Cultured cortical neurons (14-18 DIV) plated on a 96-well plate were washed with Neurobasal-A medium (Invitrogen) three times and were incubated with BDNF alone (100 ng/ml) or BDNF (100 ng/ml) plus K252a (200 nM) (Sigma-Aldrich) for appropriate time at 37 °C, followed by washing with ice-cold Neurobasal-A medium. The assay buffer comprising Neurobasal-A medium containing 120 nM [3H]muscimol (specific radioactivity, 1110.0 GBq/mmol) (PerkinElmer Life Sciences) with or without 150 µM muscimol (Sigma-Aldrich) was added to each well. The plate was incubated for 30 min on ice, followed by three quick washes with ice-cold neurobasal-A medium. Hundred microliters of liquid scintillator (MicroScint Mixture-20; PerkinElmer Life Sciences) was added to each well, and the radioactivity was counted on a TopCount NXT (PerkinElmer Life Sciences). Nonspecific binding in the presence of 150 µM muscimol (40-80 dpm) was subtracted from that in its absence (500-780 dpm) to yield the specific binding. Each assay was done in triplicate.


Figure 1
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  		FIGURE 1.
GABA-evoked whole cell currents in response to BDNF application in WT and PRIP-DKO hippocampal neurons. BDNF (5 ng/ml) was applied for 10 min as indicated by a solid bar, and the amplitude of GABAA receptor response elicited by application of GABA (10 µM) was measured every 2.5 min. Cultured hippocampal pyramidal neurons (14-18 DIV) of both genotypes (WT, filled circle (n = 3); PRIP-DKO, filled square (n = 4)) were used. The results were represented as the means ± S.E. Each result of WT and PRIP-DKO was statistically compared as indicated by * and ** for p < 0.05 and p < 0.01, respectively (Student's t test).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of GABAA Receptor Currents by BDNF in Hippocampal Neurons Isolated from WT and PRIP-DKO Mice—The precise molecular mechanisms that underlie BDNF-mediated alterations in GABAA receptor activity remain unclear. We have previously reported that PRIP-1 plays an important role in the modulation of GABAA receptor function by regulating the receptor trafficking (19) and phosphorylation (17). To test whether PRIP proteins contribute to the observed BDNF-dependent alterations of GABAA receptor activity (2-5), we have carried out experiments using PRIP-DKO mice, because PRIP-DKO mice became available recently as described above.

We first examined whether BDNF alters GABA responses in PRIP-DKO hippocampal neurons by measuring the whole cell current responses. Because presynaptic effects of BDNF on inhibitory synaptic transmission have been reported (29), we measured GABA-evoked Cl- currents (IGABA). Hippocampal neurons cultured from both WT and PRIP-DKO neonatal mice (P0 or P1) were analyzed by voltage clamp recordings. The application of 10 µM GABA to neurons from both genotypes induced a GABAA receptor-mediated inward current with a similar amplitude, which was completely blocked by 10 µM bicuculline (n = 3 each genotype; data not shown). In agreement with previous findings (2-5), the initial transient increase in the GABA-evoked currents within 2.5 min of BDNF (5 ng/ml) application in WT neurons (n = 3) was followed by a prominent decrease within 10 min (Fig. 1). These effects were blocked in the presence of 200 nM of K252a, a broad tyrosine kinase inhibitor commonly used to demonstrate involvement of signaling via TrkB receptors (data not shown). The inhibition of GABA-evoked current amplitude continued for ~30 min, reaching 55 ± 14% of the initial control levels. Strikingly, the amplitude of IGABA in neurons from PRIP-DKO mice did not exhibit a decrease upon BDNF application but instead exhibited a gradual increase reaching 145 ± 27% of control over the same time course (Fig. 1). Similar differences in BDNF-mediated alterations in GABA-evoked current were also obtained in recordings from acutely dissociated P14 hippocampal CA1 pyramidal neurons (25) from either WT or PRIP-DKO mice (results not shown).

Studies published previously have provided evidence for two possible mechanisms underlying BDNF-dependent regulation of GABAA receptor currents that include alterations in cell surface expression and/or phosphorylation of GABAA receptor beta3 subunit (3-5). Given that GABAA receptor beta subunits directly associate with AP2 proteins of endocytic protein machinery in a phosphorylation-dependent manner (7), the two proposed mechanisms may in fact act in a coordinated fashion to mediate the BDNF-dependent regulation of GABAA receptors. We tested this hypothesis by examining both phosphorylation of the beta3 subunit and its expression at the neuronal cell surface in PRIP-DKO mice in response to BDNF.


Figure 2
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  		FIGURE 2.
BDNF-induced phosphorylation of GABAA receptorbeta3 subunit. A, time course of BDNF-activated phosphorylation of beta3 subunit in cortical neurons (14-18 DIV). Cultured neurons of WT (left panels) or PRIP-DKO (right panels) mice were incubated in the presence of BDNF (100 ng/ml) for 5, 15, or 30 min, followed by immunoblot analysis. The upper and lower panels represent blots obtained using anti-P-beta3 antibody and anti-beta2/3 antibody, respectively. The graph shows the summary of the results (n = 3) analyzed using Image Gauge software (Fujifilm), represented as the percentage of the control at each time. The data are represented as mean ± S.E. (Student's t test; *, p < 0.05; **, p < 0.01). B, comparison of protein expression levels in the cortex of WT and PRIP-DKO mice. Cultured cortical neurons (21 DIV) of both genotypes were lysed, and proteins were resolved using SDS-PAGE, followed by immunoblotting with anti-PRIP-1, anti-PRIP-2, anti-TrkB, anti-pan PKC, anti-PP1, anti-PP2A, and anti-beta-tubulin antibody. The left panel shows the typical blot, and the right graph shows the results represented as the percentages of WT expression (n = 3). The standard protein markers are indicated.

 
Regulation of GABAA Receptor beta3 Subunit Phosphorylation by BDNF in PRIP-DKO Neurons—Activation of BDNF/TrkB receptor-dependent signaling pathways leads to an increase in PKC activity and PKC-dependent phosphorylation of serines 408 and 409 (Ser408/Ser409) in the large intracellular loop of the GABAA receptor beta3 subunit, followed by a rapid dephosphorylation by PP2A, thus causing a biphasic change in the phosphorylation state of these receptors (4). We have previously reported that PRIP-1 binds to and inactivates another phosphatase, PP1c (16), as well as GABAA receptor beta3 subunit (17) and regulates beta3 subunit phosphorylation at these same residues mediated by protein kinase A (17). Therefore, PRIP proteins may play an important role in the regulation of GABAA receptor phosphorylation in response to BDNF.

To test this hypothesis, we examined whether BDNF-mediated changes in phosphorylation levels of the beta3 subunit were altered in cortical neurons from PRIP-DKO mice (Fig. 2A). Cultured cortical neurons (14-18 DIV) of both genotypes were treated with BDNF (100 ng/ml) for 5, 15, and 30 min, and cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes Ser408 and Ser409 in their phosphorylated form (anti-P-beta3 antibody) (4). In WT neurons, BDNF-dependent biphasic modulation of the beta3 subunit phosphorylation was observed with an initial 2-3-fold increase in the phosphorylation of beta3 subunit within 5 min, followed by a decrease to 69 ± 5% of the initial control level within 30 min, similar to findings previously observed in rat neurons (4). In contrast, phosphorylation levels of the beta3 subunits in PRIP-DKO neurons exhibited a gradual increase to 155 ± 9% level of the control over the same period of time. To control for possible alterations in the expression levels of signaling proteins participating in the BDNF/TrkB receptor signaling pathway in PRIP-DKO neurons, we performed Western blotting using specific antibodies for TrkB receptor, PKC, PP1, and PP2A. Our results indicated that the levels of these proteins in cultured cortical neurons were not significantly altered in the PRIP-DKO in comparison with WT mice (Fig. 2B).

PRIP Proteins Mediate Binding of Protein Phosphatases PP1 and PP2A to GABAA Receptors—The dramatic alteration in BDNF-mediated receptor phosphorylation of the beta3 subunits in PRIP-DKO neurons suggested that PRIP proteins may be important for the regulation of phosphatase PP2A activity (4), in addition to the previously reported modulation of PP1 (17). We therefore examined the association between PRIP-1 and PP2A. Co-immunoprecipitation studies were performed with anti-Myc antibody using the cell lysates of COS7 cells expressing combinations of rPRIP-1 and Myc-tagged PP2A (Fig. 3). Because COS7 cells do not express endogenous PRIP-1 (14), the co-immunoprecipitated recombinant rPRIP-1 was only detected in lane 3, indicating the interaction between PRIP-1 and PP2A (Fig. 3A). The analysis was further performed using either control rabbit IgG or anti-PRIP-1 antibody to immunoprecipitate endogenous PRIP-1 from cultured cortical neurons (Fig. 3B). An immunoreactive band detected by anti-PP2A antibody was present in PRIP-1 immunoprecipitates from lysates of cultured cortical neuron (21 DIV) but was absent in control IgG, indicating that PRIP-1 interacts with PP2A. Further, we carried out immunoprecipitation experiments to map the region of PRIP-1 important for binding to PP2A using COS7 cells expressing Myc-tagged PP2A and C-terminal truncated mutants of rPRIP-1 (see also Fig. 5A). Three rPRIP-1 mutants (rPRIP-1 (1-929), rPRIP-1 (1-585), and rPRIP-1 (1-297)) as well as full-length rPRIP-1 were immunoprecipitated with anti-Myc antibody but not control rabbit IgG, whereas the mutant rPRIP-1 (1-82) was not co-immunoprecipitated (Fig. 3C). PP2A binds to PRIP-1 at the residues 83-297, which is different from those for beta subunit (the residues 544-568, which will be described in Fig. 5B), suggesting that the association of PP2A and beta subunits to PRIP is not competitive. To address this, the binding of PRIP, PP1, and PP2A to GABAA receptor beta subunits was also analyzed using GST-beta3 fusion protein in pulldown assay from rat brain lysates (Fig. 3D). Specific binding of PRIP-1, PRIP-2, PP1, and PP2A to GST-beta3 fusion protein was detected in brain lysates. We further examined the complex formation by immunoprecipitation assays using anti-beta2/3 antibody, followed by Western blotting with PP2A antibody. The association of PP2A with beta2/3 subunits of GABAA receptors detected in lysates from WT mice brains was reduced to a background level in lysates from PRIP-DKO mice brains (data not shown), suggesting that PRIP is important for the association of PP2A with the beta subunit of GABAA receptors.

To test whether binding of PRIP-1 to PP2A affects its activity, we used a commercially available serine/threonine phosphatase assay system for PP2A (Promega; catalog number V2460). The activity of PP2A showed no significant change in the presence of PRIP-1 even at very high concentrations of PRIP-1 (20-50 times molar excess; data not shown).


Figure 3
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  		FIGURE 3.
PRIP-1 associates with PP2A and GABAA receptor beta subunits. A and B, lysates from COS7 cells transfected with combinations of Myc-tagged PP2A/pRK5 and rPRIP-1/pSG5 (A) or from rat cultured cortical neurons (B) were immunoprecipitated (IP) by anti-Myc antibody or by anti-PRIP-1 antibody and rabbit IgG, respectively. Then immunoblotting was performed with anti-PRIP-1 antibody or anti-Myc antibody as a primary antibody (A) or with anti-PRIP-1 antibody or anti-PP2A antibody (B). C, HEK293 cells were transfected with Myc-tagged PP2A and a series of truncated rPRIP-1 (see Fig. 5A) and immunoprecipitated with anti-Myc antibody or control rabbit IgG, followed by immunoblotting with anti-PRIP-1 antibody. The arrowheads indicate a band for each rPRIP-1 mutant. The proteins were resolved using 10% (left) or 15% (right) SDS/PAGE gels. D, association among PRIP-1, PRIP-2, PP1, and PP2A assessed by GST-beta3 (residues 303-426) pulldown assay using brain lysates from WT mice. Western blot (WB) analysis was performed using indicated primary antibodies. Two or three other experiments provided similar results in each case. Standard molecular size markers are indicated.

 
Co-localization of PRIP with GABAA Receptor beta Subunits at the Cell Surface—To investigate the association of PRIP with GABAA receptors at the single cell level, we employed confocal microscopy using COS7 cells transiently co-expressing rPRIP-1/pDsRedN1 and GABAA receptor {alpha}1/beta2 subunits tagged with the 9E10 epitope at the N terminus ({alpha}1Myc and beta2Myc; Fig. 4A). Cell surface-expressed recombinant {alpha}1Myc and beta2Myc GABAA receptors were visualized with monoclonal anti-9E10 antibody, followed by staining with fluorescein isothiocyanate-conjugated anti-mouse secondary antibody under nonpermeabilized conditions (Fig. 4A, panel a). The cells in which both rPRIP-1 and {alpha}1Myc and beta2Myc GABAA receptors were expressed (Fig. 4A, panel b, dots), showed co-localization of rPRIP-1 and GABAA receptors at the surface membrane (Fig. 4A, panel c, arrowheads).

The co-localization of PRIP-1 and surface GABAA receptors in cultured cortical neurons was also examined by confocal microscopy. As shown in Fig. 4B, surface GABAA receptors were detected under nonpermeabilizing conditions using an antibody to the beta2/3 subunits of GABAA receptors, followed by detection of PRIP-1 signals (green) after permeabilization with saponin. The green signals were either merged (61.2 ± 0.9%), attached with (9.5 ± 0.8%), or detached (29.5 ± 0.7%) from the red signals specific for the beta2/3 subunits, indicating that a significant amount of PRIP-1 (~70%) was co-localized with the cell surface-expressed beta2/3 subunit of GABAA receptors under these conditions.


Figure 4
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  		FIGURE 4.
Co-localization of PRIP-1 and GABAA receptors at the plasma membrane. A, rPRIP-1 (red) colocalized with recombinant {alpha}/beta-GABAA receptors (green) in COS7 cells (see "Experimental Procedures"). The dots indicate cells expressing GABAA receptors, whereas the asterisks indicate cells that did not express GABAA receptors on the cell surface (panel b). The arrowheads in the merged image (panel c) show the co-localization of rPRIP-1 and GABAA receptors. More than 15 fields in three independent experiments gave similar images. B, PRIP-1 (green) co-localizes with GABAA receptor beta2/3 subunit (red) in cortical neurons (see "Experimental Procedures"). A representative merged image and the enlargements of areas 1, 2, and 3 are shown. Arrowheads, arrows, and asterisks represent merged (yellow spot), attached, and detached puncta between the green and the red, respectively. More than 10 fields in three independent experiments gave similar images. A graph shows quantification of the three categorized signals by three unaware persons, which included more than 100 green signals from five independent fields and represents percentages of the total number of green puncta (means ± S.E. (n = 3)). Similar results were obtained using mouse anti-beta-chain antibody (Chemicon) for detection of GABAA receptors (data not shown). The bar indicates 10 µm.

 
Identification of the GABAA Receptor beta Subunit Binding Site in PRIP—To selectively disrupt the interaction between PRIP-1 and GABAA receptors, experiments were performed to determine the binding region in PRIP proteins recognized by the beta subunits. In these experiments we employed various rPRIP-1 truncation mutants constructed in pSG5 or pETHis6-30 vector (Fig. 5A). Pulldown assays were performed with a GST fusion protein of the intracellular domain of the GABAA receptor beta1 subunit (residues 303-426). As shown in Fig. 5B, two constructs of rPRIP-1 with C-terminal deletions (amino acid residues 1-929 (100 kDa) and 1-585 (66 kDa)) were able to efficiently bind to the beta1 subunit as well as full-length rPRIP-1 (130 kDa). In addition, three constructs encoding truncations of the PRIP-1 N terminus (residues 399-1096 (79 kDa), 465-1096 (72 kDa), and 544-1096 (62 kDa)) were also able to bind to the beta1 subunit. In contrast rPRIP-1(569-1096) mutant, which has a further deletion of 25 amino acids (compared with rPRIP residues 544-1096), could no longer bind GST-beta1. Thus, residues 544-568, located in the intermediate region between X and Y domains of rPRIP-1, are important for the association with the beta1 subunits of GABAA receptors. As shown in the Fig. 5A (bottom sequence alignment), a highly conserved acidic amino acid cluster is found in this PRIP-1 region. In addition, this region is highly conserved in PRIP-2, indicating that PRIP-2 is also likely to bind to beta subunits. In agreement with this, GST-beta1 was also shown to interact with hPRIP-2 (residues 597-1154). The hPRIP-2 was coprecipitated with beta1 subunit as well as rPRIP-1(544-1096) (Fig. 5C). Finally, using GST pulldown assays, we confirmed that hPRIP-2 has the ability to bind to all beta subunits but not to {alpha}1 subunit (Fig. 5D).

To test whether the association between PRIP proteins and GABAA receptor beta subunits is implicated in the membrane localization of PRIP-1, the highly conserved acidic amino acid cluster of residues in rPRIP-1 (PRIP-1 binding peptide, residues 553-565; see Fig. 5A), was introduced into the pIRES2-EGFP vector to visualize the cells transfected with the plasmid. HEK293 cells were co-transfected with the PRIP-1-binding peptide plasmid, the {alpha}1Myc and beta2Myc subunits of GABAA receptor, and rPRIP-1/pDsRedN1, followed by analysis by confocal microscopy. Fig. 5E shows the cells in which the PRIP-1-binding peptide was not expressed (panels a-c) or highly expressed (panels d-f) as judged by green fluorescent protein fluorescence intensity (panels a and d, respectively). Cells without PRIP-1-binding peptide showed the co-localization of GABAA receptor and PRIP-1 at the plasma membrane (Fig. 5E, b/c-merged panel), like that seen in Fig. 4A (panel c), whereas the cells that highly co-expressed the binding peptide exhibited a cytoplasmic distribution of PRIP-1 (Fig. 5E, panel f), thus showing little co-localization (e/f-merged), indicating that the residues 553-565 are important for the targeting of PRIP-1 to the plasma membrane.


Figure 5
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  		FIGURE 5.
Mapping analysis of PRIP association with beta subunits of GABAA receptors. A, schematic representation of a series of truncated rPRIP-1 and hPRIP-2 constructs (top) and conservation of amino acids of X-Y intermediate region between PRIP-1 and PRIP-2 (bottom). The numbers indicate amino acid residues and digestion sites by restriction enzyme. PH, a pleckstrin homology domain; EF hand, an EF hand-like motif; X and Y, distorted triose phosphate isomerase barrel-like domains; C2, a C2 domain; D, an assumed rigid domain structure (10). A summary of the binding to beta subunits or PP2A is shown in the right side as + (bound) or -(unbound). The two-headed arrow at the bottom represents the region used to synthesize the beta subunit binding peptide corresponding to the binding site in PRIP proteins. B, GST pulldown assays. A series of truncated rPRIP-1 were incubated with GST-beta1 (residues 303-426), followed by the immunoblotting with streptavidin-horseradish peroxidase. An arrowhead indicates rPRIP-1 bound to GST-beta1. C, GST-beta1 (residues 303-426) pull down assay was performed with rPRIP-1 (residues 544-1096) (lane 1) and hPRIP-2 (residues 597-1154) (lane 2). An arrowhead indicates bound PRIP, and a band seen below in lane 2 appears to be a degraded hPRIP-2 (residues 597-1154). D, hPRIP-2(597-1154) bound to GST-beta1-3 but not to GST-{alpha}1. Two or three other experiments provided similar results (B-D). Standard molecular size markers are indicated. E, PRIP-1-binding peptide plasmid (green; panels a and d) inhibits the localization of PRIP (red; panels c and f) with GABAA receptor (blue; panels b and e) at the plasma membrane in HEK293 cells. A merged image between panels b and c or between panels e and panels f is shown on the far right, respectively. Typical images are shown. More than 10 fields in two independent experiments gave similar images.

 
Together these results suggest that PRIP proteins specifically interact with GABAA receptor beta subunits as well as PP2A, thus regulating the recruitment of PP2A to GABAA receptors expressed at the cell surface to effect their dephosphorylation at serine residues Ser408/Ser409, in a fashion similar to that already described for association with PP1 (17).

Changes in Cell Surface GABAA Receptor Number in WT and PRIP-DKO Neurons in Response to BDNF Application—To elucidate the correlation between the phosphorylation of beta subunit and surface receptor numbers, we examined whether levels of surface expressed receptors were altered in response to BDNF (100 ng/ml) by [3H]muscimol radioligand binding assay using cultured cortical neurons (30). In WT neurons, [3H]muscimol binding revealed a BDNF-dependent increase in GABAA receptors expressed as 118 ± 8% of control at 5 min, followed by a prominent decrease to 74 ± 8% at 10 min and 84 ± 7% of control at 25 min (Fig. 6, WT). The decrease observed at 10 min after the addition of BDNF was blocked by K252a, suggesting the dependence on TrkB receptor activation. In contrast, application of BDNF to PRIP-DKO neurons did not cause a decrease in surface GABAA receptor levels but instead resulted in a small increase to 113 ± 9% at 25 min (Fig. 6, PRIP-DKO). Therefore, it is likely that PRIP-mediated alterations in GABAA receptor surface stability may partly be attributed to the phosphorylation status of the receptor beta subunits, which inhibits the association with AP2 proteins leading to the inhibition of clathrin-dependent receptor internalization (7).

PRIP-binding Peptide Blocks the IGABA Attenuation Induced by BDNF in WT Hippocampal Neuron—To analyze the functional consequences of interaction between PRIP and beta subunits in neurons, we examined whether the synthetic peptide (amino acid residues 553-566), which mimics the receptor binding region of rPRIP-1 (Fig. 5A), blocks the BDNF-mediated GABAA receptor functional modulation. The peptide and its scrambled version were used for the measurement of GABA-evoked Cl- currents in cultured rat hippocampal neuron (14-17 DIV; Fig. 7A) or freshly dissociated WT mouse hippocampal neurons prepared from P13-15 (Fig. 7B). In both assays, the attenuation of IGABA induced by BDNF seen in the WT neurons was blocked by the addition of the binding peptide to the patch pipette solution but not its scrambled version. In the presence of the binding peptide, IGABA showed an increase in response to BDNF in a way similar to those observed in PRIP-DKO mice (Fig. 1).


Figure 6
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  		FIGURE 6.
Regulation of GABAA receptor surface levels by BDNF in WT and PRIP-DKO mice. Cultured cortical neurons (14-18 DIV) were incubated in the absence (-) or presence (+) of 100 ng/ml BDNF for 5, 10, or 25 min or together with 200 nM K252a for 10 min. The changes of cell surface GABAA receptors were assessed by a [3H]muscimol binding assay (see "Experimental Procedures"). The summary of the specific binding is represented as the percentage of the control at each time. The data are presented as the means ± S.E. (Student's t test; *, p < 0.05; WT, n = 4; PRIP-DKO, n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Modulation of GABAergic inhibition by BDNF may play a critical role in the regulation of the inhibitory synaptogenesis, as well as overall neuronal activity in the central nervous system. It is therefore of interest to understand fully the molecular mechanisms underlying the BDNF-induced modulation of GABAA receptor activity at inhibitory synapses. Evidences accumulated so far have indicated that BDNF, acting via the TrkB receptor, modulates both GABAA receptor phosphorylation (4) and cell surface expression (3, 5), two processes that may be functionally related (7). On the basis of the already established role of PRIP proteins in the regulation of PP1 (16, 17, 22), we hypothesized that a similar role can be attributed to PRIP proteins with respect to PP2A, a phosphatase shown to mediate dephosphorylation of the beta3 subunit in response to BDNF (4). Therefore, in this study, using biochemical, electrophysiological, and genetic approaches, we demonstrate a critical role for PRIP proteins as regulators of protein phosphatases 1 and 2A in the postsynaptic modulation of GABAA receptor activity by BDNF. Whole cell recordings revealed that in neurons from WT mice, BDNF application caused a reduction of IGABA with a transient initial increase similar to that reported in several previous studies (2-5). In contrast, in neurons obtained from PRIP-DKO mice, BDNF application did not cause a decrease in IGABA but rather caused a slight increase in GABAA receptor currents, suggesting a markedly altered signaling response of GABAA receptors to BDNF in the absence of PRIP proteins.


Figure 7
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  		FIGURE 7.
PRIP-binding peptide alters the regulation of GABA-evoked whole cell currents by BDNF. GABA (10 µM)-activated membrane currents (whole cell recording) were recorded using WT cultured rat hippocampal neuron (14-17 DIV) (A, n = 3 for each peptide) or freshly dissociated mouse hippocampal neuron from 13-15-day-old C57BL/6J mice (WT) (B, n = 3 for each peptide) at a holding potential of -50 mV. The patch pipette contained either binding peptide (open circle) or its scrambled version (closed circle)at3 µg/ml. The results were represented as the means ± S.E. for each series of experiment. The data were statistically compared with the control (scrambled peptide) as indicated by * and ** for p < 0.05 and p < 0.01, respectively (Student's t test). The solid bars indicate the BDNF (20 ng/ml) application.

 
We attempted to establish the molecular mechanisms that may underlie PRIP protein-dependent modulation of GABAA receptor function by BDNF. We found no change in the expression levels of molecules that have been previously established to mediate the BDNF-dependent regulation of GABAA receptor activity (4), including TrkB, PKC, PP1, and PP2A, in the absence of PRIP proteins. However, we observed a dramatically altered response to BDNF application of GABAA receptor phosphorylation in neurons from PRIP-DKO in comparison with WT mice. In neurons from WT mice, BDNF application caused a biphasic change in phosphorylation with a rapid 2-3-fold increase in phosphorylation of Ser408/Ser409 in GABAA receptor beta3 subunits, followed by dephosphorylation (to 67% of control within 30 min) similar to the previously described effects of BDNF on GABAA receptor beta3 subunit phosphorylation in rat hippocampal and cortical neurons (4). In contrast, in neurons from mice lacking PRIP proteins, GABAA receptor beta3 subunit phosphorylation upon BDNF application was dramatically altered, showing only a slow sustained increase in the phosphorylation of the receptor to 155% of control within 30 min. These results suggest that PRIP may play an important role in controlling GABAA receptor dephosphorylation by PP2A downstream of TrkB receptors activated by BDNF.

PRIP-1 plays an important regulatory role in targeting of PP1c to GABAA receptors beta subunits (17). We hypothesized that PRIP-1/2 (PRIP-1 and -2) may have a similar regulatory role in targeting of PP2A to GABAA receptor beta subunits to coordinate the receptor dephosphorylation downstream of BDNF-dependent signaling (4). Importantly, we found that PRIP-1/2 interact directly with PP2A, and the interaction is not competitive for beta subunits. Consistent with these observations, the amount of PP2A immunoprecipitated with anti-beta2/3 antibody was reduced in the PRIP-DKO brain, suggesting that PRIP proteins are scaffolding and/or regulatory molecules important for the recruitment of phosphatases, PP1c, and PP2A to GABAA receptors.

In addition to an altered response of GABAA receptor beta3 subunit phosphorylation in the presence of BDNF, we also detected alterations in the trafficking of GABAA receptors in the presence of BDNF in PRIP-1/2-deficient mice. Whereas in WT neurons BDNF caused an initial up-regulation of the surface expressed GABAA receptors to 118% followed by the receptor down-regulation to 84% within 25 min, in PRIP-DKO neurons only a slow sustained increase in surface receptor numbers was observed over the same period of time. Therefore, PRIP proteins, perhaps in the complex with PP2A and PP1, may also be important for regulating the stability of GABAA receptors at the neuronal cell surface. The alterations in surface GABAA receptor levels appeared to correlate well with the observed changes in the receptor phosphorylation and IGABA in PRIP-DKO neurons, in agreement with the recent observation that phosphorylation of GABAA receptors can inhibit their internalization by blocking the interaction with AP2 adaptor proteins (7).

Our experiments have demonstrated that PRIP-1/2 partially co-localize with the internalized pool of GABAA receptors when expressed in HEK293 cells, as assessed by a constitutive receptor endocytosis assay (28). Furthermore, we have very recently elucidated the formation of a complex including PRIP-1, clathrin, AP2 proteins (beta2 and µ2 subunits), and protein phosphatases (PP1 and PP2A) in rat and mouse brain using immunoprecipitation experiments with anti-PRIP-1 antibody.5 It suggests that PRIP-1/2 proteins and phosphatase complexes are associated with clathrin-coated vesicles, thereby playing a role in phospho-dependent endocytosis of GABAA receptors regulated by clathrin/AP2 (7, 31).

The effects of BDNF on GABAA receptor surface levels and endocytosis appear complex. Using antibody labeling and immunofluorescence approaches, BDNF was found to promote the receptor internalization using antibodies specific for {alpha}2 subunit in cultured mouse cerebellar granule cells (7-10 DIV prepared from P5-6) (5) or beta2/3 subunits in cultured rat hippocampal neurons (20 DIV prepared from embryonic day 18) (3). However, a BDNF-dependent increase in surface GABAA receptors containing beta3 subunits has been established using biotinylation approaches followed by immunoblotting with anti-beta3-specific antibodies in cultured rat hippocampal and cortical neurons (8-14 DIV prepared from embryonic day 17) (4). In the current study, using a radio-ligand binding approach to monitor the surface levels of {alpha}/beta subunits in cultured mouse cortical neurons (14-18 DIV prepared from P0-1), we observed an increase in GABAA receptor levels at the cell surface within 5 min, followed by a prominent decrease at later time points. It is difficult to explain the discrepancy between these studies clearly, but there are some possibilities. Because cell surface stability of GABAA receptors is regulated by many molecules interacting with specific subunit(s) of GABAA receptors (32), it is possible that BDNF causes different effects depending on the predominant receptor subtype(s) expressed. Also, the age of neuronal cultures in vitro may also influence the final effect of BDNF, given that GABAergic transmission is altered from excitatory to inhibitory actions during early developmental period (33). Thus, the variability of BDNF actions may depend on several factors including the methodology used, the neuronal cell types, neuronal ages, and the species from which the neurons were derived.

Similarly, the effects of activation of kinases and phosphatases on GABAA receptor surface stability and endocytosis are also complex, adding another level of variability of the effects observed upon BDNF application. Consistently, it has been shown that in recombinant systems, internalization of GABAA receptors is enhanced by the activation of PKC (28, 34-38). However, these effects are independent of direct phosphorylation of receptor subunits (35), and it is currently unclear whether a similar PKC-mediated down-modulation of surface GABAA receptors exists in all neuron subtypes.

The lack of dephosphorylation of the GABAA receptor in PRIP-1/2-deficient neurons is likely due to an altered recruitment or activity of protein phosphatases PP1 and/or PP2A to the receptor. In contrast, the loss of the rapid BDNF-dependent phosphorylation of the receptor at early time points is currently unclear. A previous report has indicated that BDNF-dependent activation and targeting of PKC to GABAA receptor beta3 subunits results in an increase in GABAA receptor phosphorylation at serine residues 408/409 (4). Therefore, it is possible that the recruitment of PKC to the receptor may also be altered in the absence of PRIP-1/2, although the expression levels of PKC remain unchanged. However, it is important to notice that some PKC isoforms, such as PKCbetaII, were shown to bind directly to the beta subunits of GABAA receptors (27). Alternatively, the lack of the robust increase in phosphorylation in PRIP-DKO neurons in response to BDNF may be due to the increased activity of PP1 in the cytoplasm, given that the activity of PP1 in PRIP-1 KO mice was enhanced by 43% (17).

In conclusion, the acute exposure to BDNF leads to a robust phosphorylation of the GABAA receptor beta3 subunit followed by a rapid dephosphorylation, thus causing a decrease in inhibitory synaptic transmission. The decrease in receptor phosphorylation is mirrored by the facilitation of GABAA receptor internalization, consistent with the recent observation that the direct phosphorylation of receptor beta subunits inhibits receptor endocytosis (7). However, BDNF-dependent modulation appears strikingly altered in PRIP-1/2 null mice, and we demonstrate here that this difference may result from inappropriate association of phosphatases PP1 and PP2A with GABAA receptor beta subunits. The present study demonstrates that PRIP proteins play a key role in dephosphorylation of GABAA receptors by PP1 and PP2A, thereby affecting the levels of GABAA receptor activity and stability at the cell surface. In addition, dissecting the role of PRIP proteins in BDNF-dependent regulation of GABAergic inhibition provides further insights into mechanisms underlying formation and maintenance of inhibitory synapses.


   FOOTNOTES
 
* This work was supported by a Grant-in-Aid for Scientific Research from the MEXT of Japan (to T. K., Y. M., A. K., and M. H.) and by funds from the Cooperative Study Program of National Institute for Physiological Sciences (to T. K., J. N., and M. H.), the Epilepsy Research Foundation (to M. H.), the Kato Memorial Bioscience Foundation (to T. K.), the Naito Foundation (to T. K.), and the Takeda Science Foundation (to T. K.). The costs of publication of this article were 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. Back

1 Japan Society for the Promotion of Science research fellow. Back

2 Japan Society for the Promotion of Science research fellow. Recipient of the Iwadare Scholarship. Back

3 To whom correspondence should be addressed. Tel.: 81-92-642-6317; Fax: 81-92-642-6322; E-mail: hirata1{at}dent.kyushu-u.ac.jp.

4 The abbreviations used are: BDNF, brain-derived neurotrophic factor; GABA, {gamma}-aminobutyric acid; PP, protein phosphatase; PRIP, phospholipase C-related but catalytically inactive protein; DKO, double knock-out; WT, wild type; Pn, postnatal day n; DIV, days in vitro; GST, glutathione S-transferase; PKC, protein kinase C; PIPES, 1,4-piperazinediethanesulfonic acid. Back

5 T. Kanematsu, M. Fujii, A. Kuratani, J. T. Kittler, J. Nabekura, S. J. Moss, and M. Hirata, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Makiko Hirata for technical assistance.



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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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A. Mizokami, T. Kanematsu, H. Ishibashi, T. Yamaguchi, I. Tanida, K. Takenaka, K. I. Nakayama, K. Fukami, T. Takenawa, E. Kominami, et al.
Phosholipase C-Related Inactive Protein Is Involved in Trafficking of {gamma}2 Subunit-Containing GABAA Receptors to the Cell Surface
J. Neurosci., February 14, 2007; 27(7): 1692 - 1701.
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