GABAC-Receptor Stimulation Activates cAMP-Dependent Protein Kinase via A-Kinase Anchoring Protein 220

Journal of Pharmacological Sciences

J Pharmacol Sci 106, 578 – 584 (2008)4

©2008 The Japanese Pharmacological Society

Full Paper

GABAC-Receptor Stimulation Activates cAMP-Dependent Protein Kinase via A-Kinase Anchoring Protein 220 Li Yang1,#, Yasuhisa Nakayama1,*, Naoki Hattori1, Bing Liu1, and Chiyoko Inagaki1 1

Department of Pharmacology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan

Received August 20, 2007; Accepted February 5, 2008

Abstract. In our previous study, anti-apoptotic effects of GABAC-receptor stimulation was suppressed by inhibitors of cAMP-dependent protein kinase (PKA), implying GABAC receptor– mediated PKA activation. The present study showed that GABAC-receptor stimulation with its agonist, cis-4-aminocrotonic acid (CACA), protected cultured hippocampal neurons from amyloid β 25 – 35 (Aβ25 – 35) peptide–enhanced glutamate neurotoxicity. This protective effect of CACA was blocked by PKA inhibitors, KT 5720 and H-89, as well as a specific GABACreceptor antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA). To test the possibility of GABAC receptor–mediated PKA activation, association of GABAC receptor with A-kinase anchoring proteins (AKAPs) and effect of an AKAP antisense oligonucleotide on the PKA activation were examined in primary cultured rat hippocampal neurons. Stimulation of the cells with CACA-activated PKA was assessed by the phosphorylated PKA substrate (135 kDa) level. Specific antibodies raised against GABAC-receptor ρ subunits precipitated each ρ subunit, AKAP220, and PKA regulatory and catalytic subunits from rat brain lysates, suggesting that ρ is associated with the AKAP220/ PKA complex. Furthermore, antisense oligonucleotide of AKAP220 suppressed such GABAC stimulation–induced PKA activation, suggesting that GABAC-receptor stimulation activates PKA via AKAP220. Keywords: GABAC receptor, cAMP-dependent protein kinase (PKA), A-kinase anchoring protein 220 (AKAP220) dent protein kinase (PKA) activation. PKA is a serine / threonine kinase with broad specificity whose fidelity in signaling is maintained through interaction with Akinase (PKA) anchoring proteins (AKAPs) (8, 9). We therefore wondered whether ionotropic GABAC-receptor stimulation activates PKA through such an anchoring mechanism. In the present study, we examined the association of GABAC-receptor ρ subunits with neuronal AKAPs, such as AKAP220, AKAP150, and microtubule-associated protein MAP2B (9 – 13), as well as GABAC receptor stimulation–induced PKA activation by monitoring phospho-PKA substrates in the rat brain neurons.

Introduction The GABAC receptor, a pentameric protein complex composed of ρ1, ρ2, and / or ρ3 subunits, is known to function as a ligand-gated chloride channel (1 – 3). The expressions of ρ1, ρ2, and ρ3 subunits and ligand-gated currents of GABAC receptor have been shown mainly in the retina. However, there have been findings that outside of retina, the expressions of GABAC receptors are observed substantially in various brain areas including the hippocampus (4 – 7). In our previous reports, the GABAC-receptor agonist cis-4-aminocrotonic acid (CACA) protected against ammoniainduced apoptotic cell death in primary cultured rat hippocampal neurons probably through cAMP-depen-

Materials and Methods

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Current address: Department of Pharmacology, School of Pharmacy, The University of Reading, Reading, UK *Corresponding author. [email protected] Published online in J-STAGE on April 3, 2008 (in advance) doi: 10.1254/jphs.FP0071362

Preparation of antibodies Anti-ρ1, -ρ2, and -ρ3 antibodies were prepared by immunizing rabbits with albumin-MBS [N-(mmaleimidobenzoyloxy) succinimide] coupled synthetic

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peptides (ρ1, NH2-AESTVHWPGREVHEPC-COOH; ρ2, NH2-MALVESRKPRRKRWTGHLEC-COOH; ρ3, NH2-TYTWITLMLDASAVKEPHQC-COOH) corresponding to amino acid sequences of rat ρ1(16 – 30) (14, 15), ρ2(15 – 33) (16), and ρ3(11 – 29) (17), respectively. Titer of antibodies was tested with Western blotting (the optimal dilution: ρ1, 1:1000; ρ2, 1:250; ρ3, 1:2000). Immunocytochemical staining Immunocytochemistry was carried out with primary cultured rat hippocampal neurons as described previously (6). Fixed cells were incubated with rabbit antiρ1, -ρ2, or -ρ3 antibody (1:200; 1:50; 1:500) and mouse anti-AKAP220 (1:50) or anti-PKARIIβ antibody (1:200) (PKA regulatory subunit type IIβ; BD Biosciences Pharmingen, San Diego, CA, USA) at 4°C overnight. The primary antibodies were visualized with respective secondary antibodies, fluorescein-conjugated anti-rabbit IgG (1:1000; ICN Pharmaceuticals, Aurora, OH, USA), and rhodamine red–conjugated anti-mouse IgG (1:2000; Molecular Probes, Eugene, OR, USA). A bibenzimidazole dye, Hoechst 33258 (10 µg/ ml; Sigma, St. Louis, MO, USA), and anti-neurofilament H monoclonal antibody (1:200; Chemicon, Temecula, CA, USA) was used for analysis of neuronal apoptosis as described previously (6). Condensed, crescent-aggregated, segmented, or fragmented nuclei were taken as apoptotic nuclei. Immunostaining was analyzed by using a fluorescence microscope (model BX50; Olympus, Tokyo) connected to cooled CCD image analyzer (Sekitech, Tokyo). More than 150 neurons for each treatment from four or five independent triplicate experiments were analyzed. Immunoprecipitation and Western blotting Adult Wistar rat hippocampi were homogenized with 0.25 M sucrose, 12.5 mM Tris / Mes (2-morpholinoethanesulfonic acid) (pH 7.4) and 1 mM EDTA / Tris (pH 7.4) containing protease and phosphatase inhibitors [10 µg / ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na3VO4]. An aliquot of protein sample (750 µg) was incubated with 0.25 µg anti-rabbit IgG and 30 µl (50% v / v) protein A Sepharose (Amersham Biosciences AB, Uppsla, Sweden) at 4°C for 45 min to avoid non-specific binding. Supernatant was incubated with primary antibody against ρ1 (1:50), ρ2 (1:50), or ρ3 (1:50) at 4°C overnight and then with 30 µl (50% v / v) protein A Sepharose for 2 h. Pellets were washed with a buffer (0.5% triton-X 100, 150 mM NaCl, 10 mM Tris / HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 10 µg/ ml aprotinin, 1 mM PMSF, and 1 mM Na3VO4) for 4 times and resuspended in

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2 × SDS (sodium dodecyl sulphate) buffer (0.125 M Tris / HCl pH 6.8, 4% SDS, 20% glycerol, and 0.05% bromphenol blue) mixed with 5% 2-mercaptoethanol with 5 min boiling. Supernatant was loaded to 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and the electrophoresed proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Bedford, MA, USA). The membrane was blocked with 5% skim milk (Difco, Sparks, MD, USA) followed by Western blotting with antibodies against PKAR1 (1:250) (PKA regulatory subunit type I), PKARIIβ (1:1000), PKAC (1:1000) (PKA catalytic subunit), AKAP220 (1:250), and MAP2B (1:1000), which were purchased from BD Biosciences Pharmingen or AKAP150 antibody (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as directed in the manufacturer’s instructions. Cell culture and drug treatment Primary neuronal cultures were prepared from hippocampi of embryonic 18-day Wistar rat fetuses as described previously (6). The cells were seeded in polyL-lysine-coated plastic dishes. Two days after seeding, the cells were exposed to 7.5 µM adenine-9β-arabinofuranoside (Ara-A) in modified Eagle’s medium (MEM) supplemented with 2 mM L-glutamine and 5% horse serum for 4 days. Cells at 8th – 11th day of culture were used. For immunocytochemical staining for apoptosis, the GABAC-receptor agonist CACA (200 µM, Sigma) was applied for 15 min before Aβ25 – 35 treatment. Aβ25 – 35 and other reagents were applied for 2 days from the 8th day of culture. The cells were exposed to glutamate (10 µM, 10 min) on the 10th day of culture and stained for identified apoptotic cells after another 2day culture in the media used during the 8th – 10th day of culture with or without Aβ and other reagents. For Western blot analysis, cells were treated for 2 min with 200 µM CACA. A specific antagonist of the GABAC receptor, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA, 15 µM; Tocris, Avonmouth, UK) or a specific inhibitor of PKA, KT5720 (1 µM; Calbiochem, La Jolla, CA, USA) or H89 (1 µM; Seikagaku, Tokyo), was added to the culture medium 1 h before CACA treatment. Cells were lysed with a lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaF, 100 mM Na4P2O7, 1% Triton-X 100, and protease and phosphatase inhibitors (10 µg/ml aprotinin, 1 mM PMSF, and 1 mM Na3VO4). Antisense oligonucleotide of AKAP220 treatment in primary rat hippocampal cell culture Antisense and sense oligonucleotides corresponding

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to the codon region (8123 – 8143) of rat AKAP220 mRNA were purchased (15). The antisense sequence was 5'-AGCGACTATCTTCTCTGCAAG-3' and the sense sequence was 5'-CTTGCAGAGAAGATAGTC GCT-3'. Transfection was performed in a serum- and antibiotics-free growth medium as described previously (6). Oligofectamine (Invitrogen, Grand Island, NY, USA) was incubated in the medium for 10 min at room temperature and further incubated with diluted oligonucleotide for 20 min. Then the mixture of oligonucleotide and oligofectamine was added to cultures and incubated overnight at 37°C with 5% CO2. A serumcontaining medium was applied to the culture to adjust it to a normal condition. Three-day–transfected cells were exposed to 200 µM CACA for 2 min and harvested immediately with the lysis buffer described above. A membrane-permeable PKA activator, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate sodium salt (pCPT-cAMP, 1 mM, 5 min; Sigma), was used to directly activate PKA. A sample of cell lysate (20 µg protein) was loaded on each lane of a 7.5% gel for SDS-PAGE, followed by Western blotting with antiphospho-(Ser / Thr) PKA substrate antibody (1:1000) (Cell Signaling Technology, Beverly, MA, USA), which does not recognize the nonphosphorylated PKA substrate motif; anti-AKAP220 antibody (1:250); or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000) (Santa Cruz Biotechnology). Densitometric analysis and statistics The density of immunoreactive bands was digitized and the integrated optical density was estimated using ImageJ (National Institutes of Health, Bethesda, MD, USA). All values were expressed as means ± S.E.M. Statistical comparisons were performed by ANOVA followed by Fisher’s PLSD test. Differences with P<0.05 were considered to be significant. Results PKA-dependent protection by GABAC receptor stimulation from Aβ/glutamate-induced neuronal apoptosis As shown in Fig. 1, Aβ/glutamate treatment obviously increased apoptotic pyramidal cell number, such an increase being prevented by co-application of 200 µM CACA with Aβ. This protective effect of CACA against Aβ/ glutamate toxicity was reversed by the GABACreceptor antagonist TPMPA at 15 µM and inhibitors of PKA, KT5720 (1 µM) and H89 (1 µM), suggesting that CACA protected the neurons from Aβ/ glutamateinduced apoptosis through stimulation of GABAC receptors and PKA activation.

Fig. 1. Effects of GABAC-receptor stimulation on Aβ/glutamateinduced apoptosis in cultured rat hippocampal neurons. A: Apoptotic neurons were assayed by double staining with Hoechst 33258 and anti-neurofilament antibody. Condensed, crescent-aggregated, segmented, or fragmented nuclei were taken as apoptotic nuclei. Arrows and arrowheads indicate pyramidal cell-like neurons and apoptotic nuclei, respectively. B: Summarized data of apoptotic neurons. Cells on the 8th day of culture were treated with Aβ25 – 35 peptide (10 nM) in the presence or absence of CACA (200 µM) for 48 h, treated with or without glutamate (10 µM) for 10 min in serumfree MEM, and then further cultured for 48 h in Aβ25 – 35 (10 nM) with or without CACA (200 µM) in growth medium. As for TPMPA, KT5720, and H89 treatment, cultures were pretreated with TPMPA (15 µM), KT5720 (1 µM), or H89 (1 µM) for 1 h before any treatments on the 8th culture day. More than 150 neurons were analyzed in each triplicate treatment for three or four experiments. *P<0.05, **P<0.01.

GABAC receptor stimulation-induced activation of PKA PKA activation induced by GABAC-receptor stimulation was examined by Western blots of phospho(Ser / Thr) PKA substrate using CACA-exposed primary cultured rat hippocampal neuronal cells (Fig. 2). Among

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Fig. 3. Association of GABAC-receptor ρ subunits with AKAPs in adult rat brain lysates. A: Anti-ρ1, -ρ2, and -ρ3 antibodies were prepared using peptides corresponding to amino acid sequences of rat ρ1(16 – 30), ρ2(15 – 33), and ρ3(11 – 29), respectively. GABACreceptor subunit ρ1 (62 kDa), ρ2 (54 kDa), and ρ3 (62 kDa) were detected by Western blotting with corresponding antibodies in adult rat brain lysates as indicated by arrows. B: Co-immunoprecipitation of AKAPs with GABAC-receptor ρ subunits in adult rat brain lysates. Immunoprecipitation was performed with anti-ρ antibodies followed by Western blotting with anti-AKAP220, -AKAP150, or -MAP2B antibody. C: Co-immunoprecipitated proteins with anti-ρ antibodies from adult rat brain lysates (750 µg) visualized by Western blotting using antibodies against AKAP220 (220 kDa), PKAR1 (48 kDa), PKARIIβ (53 kDa), or PKAC (40 kDa). IP, immunoprecipitation; L, adult rat brain lysate; N, negative control performed with nonimmunized rabbit IgG. Fig. 2. GABAC-receptor stimulation–induced PKA activation in cultured rat hippocampal neuronal cells. A: Western blot analysis of phospho-(Ser /Thr) PKA substrates (p-PKAsub). B: Summarized densitometric data of p-PKAsub135 levels (n = 5 – 7). C: Summarized densitometric data of p-PKAsub75, p-PKAsub100, and pPKAsub135 levels by CACA. *P<0.05, **P<0.01.

three bands observed at 135, 100 and 75 kDa, a 135-kDa phospho-(Ser / Thr) PKA substrate (p-PKAsub135) increased by CACA treatment (200 µM, 2 min) (Fig. 2). However, 100- and 75-kDa phospho-(Ser / Thr) PKA substrates did not increase by CACA treatment (Fig. 2C). This increase was blocked by PKA inhibitors, KT5720 (1 µM) and H89 (1 µM), and by the specific

GABAC-receptor antagonist TPMPA (15 µM), suggesting that GABAC-receptor stimulation activated PKA to phosphorylate specific substrate(s). Association of GABAC receptor ρ subunits with AKAPs Although the mechanisms of ionotropic receptormediated PKA activation are not known, possible involvement of AKAP family proteins, which transduce multiple components of signal input to PKA activation (8, 9), were examined. Since brain neurons are reportedly abundant in AKAPs such as AKAP220, AKAP150, and MAP2B (9 – 13), association of GABAC receptor and these neuronal AKAPs was tested using anti-

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GABAC-receptor antibodies. Anti-GABAC-receptor subunits ρ1, ρ2, and ρ3 antibodies raised against the specific peptide sequence for each subunit recognized protein bands with molecular size of the corresponding subunits (62, 54, and 62 kDa for ρ1, ρ2, and ρ3, respectively) on Western blotting of rat brain lysates (Fig. 3A). Immunoprecipitation using these antibodies precipitated corresponding ρ subunits as well as AKAP220, except that anti-ρ1 antibody precipitated AKAP150 as well (Fig. 3B). Thus, among the neuronal AKAPs tested, GABAC ρ subunits appeared to associate mainly with AKAP220. To test whether such a GABAC ρ subunit / AKAP220 complex is furnished with PKA, the immunoprecipitates were further blotted with antibodies against regulatory and catalytic subunits of PKA (PKAR1, PKARIIβ, and PKAC). As shown in Fig. 3C, all the GABAC-receptor ρ subunits co-immunoprecipitated PKA regulatory and catalytic subunits as well as AKAP220, suggesting the presence of the GABAC ρ subunit / AKAP220/ PKA complex. Mediation of AKAP220 in GABAC receptor stimulationinduced PKA activation Involvement of AKAP220 in signal transduction from GABAC-receptor stimulation to PKA activation was examined using the antisense oligonucleotide of AKAP220 (Fig. 4). CACA did not change the expression of AKAP220 in the sense-oligonucleotide–transfected cells (Fig. 4A). In the antisense-oligonucleotide– transfects of cultured rat hippocampal neurons, the expression of AKAP220 was reduced as compared with the sense-oligonucleotide–transfected cells (Fig. 4: A and B). CACA (200 µM, 2 min) increased phosphoPKAsub135 level in the sense-oligonucleotide–transfected cells, but not in the antisense-oligonucleotide– transfected ones (Fig. 4: C and D). Phospho-PKAsub100 or phospho-PKAsub75 level did not change with CACA treatment (Fig. 4: C and D). The membrane-permeable PKA activator pCPT-cAMP increased phosphoPKAsub100 and phospho-PKAsub75 levels, but not phospho-PKAsub135 levels, despite the transfection of either oligonucleotide (Fig. 4: C and E). Collectively, suppression of AKAP220 expression led to a loss of CACA-induced increase in phospho-PKAsub135 level, suggesting the mediation of AKAP220 between GABAC-receptor stimulation and PKA activation to phosphorylate a specific substrate. Discussion The present study evidenced that GABAC-receptor ρ subunits associate with AKAP220. Although association

Fig. 4. Effects of AKAP220 antisense oligonucleotide on GABACreceptor stimulation or a membrane-permeable PKA activatorinduced PKA activation. A and B: Densitometric analysis of AKAP220 expression in the sense- or antisense-oligonucleotide– transfected cells (n = 9, B). C: Effects of CACA and pCPT-cAMP on phospho-(Ser /Thr) PKA substrates (p-PKAsub) levels in the senseor antisense-oligonucleotide–transfected cells. D and E: Summarized densitometric data of p-PKAsub levels with CACA-treated cells (n = 9, D) and pCPT-cAMP–treated cells (n = 7, E). *P<0.05, **P<0.01.

of GABAC-receptor ρ subunits with some scaffolding proteins, such as MAP1B and ZIP3ξ (18 – 21), has been reported, metabotropic signaling of GABAC-receptor stimulation has not been demonstrated yet. Recently, several studies demonstrated that cationotropic receptor

Relation Between Rho Subunits and AKAP

stimulation regulates the effects of some kinases. Stimulation of α7 nicotinic acetylcholine receptors upregulates ERK1 / 2 phosphorylation (22). Furthermore, NMDA-receptor stimulation blocks phosphorylation of AMPA GluR1 at PKA phosphorylation site (23), and elevates phospho-ERK1/ 2 levels in cooperation with mGlu5 through the PSD-95 / Homer1b / c pathway in neurons (24). Here we showed that anionotropic GABAC-receptor stimulation up-regulates PKA activity and that this signaling is mediated by a neuronal AKAP, AKAP220. AKAPs contain a PKA-anchoring motif and a unique subcellular targeting domain to restrict its location and to tether PKA at defined locations within the cell (8, 9). Besides these features, AKAPs have additional binding sites for other signalling components such as kinases, phosphatases, or potential substrates. AKAPs coordinate signal transduction through recruiting multiple enzymes near potential substrates. AKAP220 mostly distributed in brain and testis (9, 13). Since suppression of AKAP220 expression blocked CACA-induced increase in phospho-PKAsub135 levels, AKAP220 appears to play an indispensable role in GABAC-receptor stimulation–induced phosphorylation of this PKA substrate. However, the possibility that pPKA-sub135 might be a substrate of another Ser / Thr kinase that is activated through the GABAC receptor–mediated activation of PKA cannot be negated because pCPT-cAMP did not significantly increase the pPKA-sub135 level. In contrast to this CACA-induced phosphorylation, pCPTcAMP increased phospho-PKAsub100 and phosphoPKAsub75 levels regardless of AKAP220 suppression (Fig. 4). AKAP220 thus appears to act as a transducer of GABAC-receptor stimulation to phosphorylate at least a specific PKA subtrate. Expression of AKAP220 was found to increase around dying spermatocytes after toxic stimulation in rats (14). Also, regulation of glycogen synthase kinase-3β (GSK-3β) activity by PKA and protein phosphatase 1 is reportedly mediated by AKAP220 through forming a complex (25). Since GSK3β plays a critical role in apoptosis (26), GABAC receptor / AKAP220–mediated PKA activation is probably involved in the anti-apoptotic effects of GABACreceptor stimulation. In summary, the present study provided new evidence of signaling convergence from an ionotropic GABACreceptor stimulation to PKA activation via AKAP220 to phosphorylate a specific PKA substrate, which may relate to GABAC receptor–mediated anti-apoptotic effects. Acknowledgments This work was supported by COE, High-Tech

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Research Center, Academic Frontier projects and grants from the Japanese Ministry of Education, Science, Sports, and Culture; and it was supported by the Japanese Private School Promotion Foundation. References 1 Bormann J, Feigenspan A. GABAC receptors. Trends Neurosci. 1995;18:515–519. 2 Enz R. GABAC receptors: a molecular view. Biol Chem. 2001;382:1111–1122. 3 Zhang D, Pan ZH, Awobuluyi M, Lipton A. Structure and function of GABAC receptors: a comparison of native versus recombinant receptors. Trends Pharmacol Sci. 2001;22:121– 132. 4 Boue-Grabot E, Roudbaraki M, Baxcles L, Tramu G, Bloch B, Garret M. Expression of GABA receptor ρ subunits in rat brain. J Neurochem. 1998;70:899–907. 5 Wegelius K, Pasternack M, Hitlunen JO, Rivera C, Kaila K, Saarma M, et al. Distribution of GABA receptor ρ subunit transcripts in the rat brain. Eur J Neurosci. 1998;10:350–357. 6 Yang L, Omori K, Otani H, Suzukawa J, Inagaki C. GABAC receptor agonist suppressed ammonia-induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level. J Neurochem. 2003;87:791–800. 7 Liu B, Hattori N, Zhang NY, Wu B, Yang L, Kitagawa K, et al. Anxiolytic agent, dihydrohonokiol-B, recovers amyloid beta protein-induced neurotoxicity in cultured rat hippocampal neurons. Neurosci Lett. 2005;384:44–47. 8 Dell’Acqua ML, Scott JD. Protein kinase A anchoring. J Biol Chem. 1997;272:12881–12884. 9 Michel JJC, Scott JD. AKAP mediated signal transduction. Annu Rev Pharmacol Toxicol. 2002;42:235–257. 10 Davare MA, Dong F, Rubin CS, Hell JW. The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons. J Biol Chem. 1999;274:30280–30287. 11 Ferhat L, Represa A, Ferhat W, Ben-Ari Y, Khrestchatisky M. MAP2d mRNA is expressed in identified neuronal populations in the developing and adult rat brain and its subcellular distribution differs from that of MAP2b in hippocampal neurons. Eur J Neurosci. 1998;10:161–171. 12 Lilly SM, Alvarez FJ, Tietz EI. Synaptic and subcellular localization of A-kinase anchoring protein 150 in rat hippocampal CA1 pyramidal cells: co-localization with excitatory synaptic markers. Neuroscience. 2005;134:155–163. 13 Reinton N, Collas P, Haugen TB, Skalhegg BS, Hansson V, Jahnsen T, et al. Localization of a novel human A-kinaseanchoring protein, hAKAP220, during spermatogenesis. Dev Biol. 2000;223:194–204. 14 Jindo T, Wine RN, Li LH, Chapin RE. Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid. Toxicol Pathol. 2001;29:607–616. 15 Lester LB, Coghlan VM, Nauert B, Scott JD. Cloning and characterization of a novel A-kinase anchoring protein, AKAP 220, association with testicular peroxisomes. J Biol Chem. 1996;271:9460–9465. 16 Ogurusu T, Taira H, Shingai R. Identification of GABAA receptor subunits in rat retina: cloning of the rat GABAA

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