J. Biol. Chem.-2013-Zhang-jbc M112 449462

Role of AC Anchoring by AKAP5 in Postsynaptic Signaling

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Adenylyl Cyclase Anchoring by A Kinase Anchor Protein AKAP5 (AKAP79/150) is Important for

Postsynaptic β-Adrenergic Signaling*

Mingxu Zhang1,2$, Tommaso Patriarchi2$, Ivar S. Stein1,2, Hai Qian1, Lucas Matt2, Minh Nguyen2, Yang K. Xiang2, and Johannes W. Hell1,2

1Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine University of Iowa, Iowa City, IA 52242

2Department of Pharmacology, School of Medicine University of California, Davis, CA 95615

*Running head: Role of AC anchoring by AKAP5 in Postsynaptic Signaling To whom correspondence should be addressed: Johannes W. Hell, PhD, 451 E Health Sciences Drive, Davis, CA 95616-8636. Phone: (530) 752 6540; FAX: (530) 752 7710; E-mail: [email protected]. $ Co-first authors. Keywords: adenylyl cyclase, AMPA receptors, β adrenergic receptors, Protein Kinase A, A kinase anchor protein, long-term potentiation

Background: AKAP5 is emerging as an adenylyl cyclase (AC) binding protein. Results: Knock-out of AKAP5 affects β-adrenergic postsynaptic signaling more than abrogating PKA targeting only in AKAP5 deletion mutants. Conclusion: AC anchoring by AKAP5 is critical for postsynaptic signaling via cAMP and PKA. Significance: β-adrenergic signaling, which depends on AKAP5-anchored AC, regulates synaptic transmission to augment alertness and memory. SUMMARY

Recent evidence indicates that the A kinase anchor protein AKAP5 (AKAP79/150) interacts not only with PKA but also with various adenylyl cyclase (AC) isoforms. However, the physiological relevance of AC-AKAP5 binding is largely unexplored. We now show that postsynaptic targeting of AC by AKAP5 is important for phosphorylation of the AMPA-type glutamate receptor subunit GluA1 on S845 by PKA and for synaptic plasticity. Phosphorylation of GluA1 on S845 is strongly reduced (by 70%) under basal conditions in AKAP5 KO mice but not at all in D36 mice, in which the PKA binding site of AKAP5 (i.e. the C-terminal 36 residues) has been deleted without affecting AC association with GluA1. The increase in S845 phosphorylation upon β-

adrenergic stimulation is much more severely impaired in AKAP5 KO than D36 mice. In parallel, long-term potentiation (LTP) induced by a 5 Hz/180 sec tetanus, which mimics the endogenous theta rhythm and depends on β-adrenergic stimulation, is only modestly affected in acute forebrain slices from D36 mice but completely abrogated in AKAP5 KO mice. Accordingly, anchoring of not only PKA but also AC by AKAP5 is important for regulation of postsynaptic functions and specifically AMPA receptor activity.

AKAPs link PKA to several of its key

substrates for fast, efficient, and selective phosphorylation of those targets (1,2). The speed, potency, and selectivity of signaling from the cAMP-producing ACs to the cAMP-activated PKA could be further enhanced if ACs would be part of the same signaling complexes as PKA. In fact, we showed previously that the AMPA-type glutamate receptor subunit GluA1 as well as the L-type Ca2+ channel Cav1.2 form complexes with the β2 adrenergic receptor (β2 AR) and PKA that also contain one or more AC isoforms along with the AC stimulating trimeric Gs protein (3-5) (see also (6) and Fig. 1). These interactions result in highly localized phosphorylation and regulation of Cav1.2 and GluA1 (3-5). Whereas structural and functional aspects of PKA anchoring by AKAPs are well established, how ACs are linked to these

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.449462The latest version is at JBC Papers in Press. Published on May 6, 2013 as Manuscript M112.449462

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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and other signaling complexes is largely unknown. Initial work showed that AC5 and AC6 can associate with the AKAP5/PKA and the AKAP6 (mAKAP)/PKA complexes for down regulation of AC activity upon stimulation of phosphorylation of the AC by PKA within these complexes establishing an important negative feedback mechanism for AC - PKA signaling (7,8). Similarly the AKAP yotiao can interact with several ACs and this interaction per se results in inhibition of AC2 and AC3 but not AC1 and AC9 catalytic activity (9). More recent work indicates that AKAP5 can interact not only with AC5 and AC6 but also AC2, AC3, AC8, and AC9 (10-12) and that AKAP5 can recruit ACs to AMPA receptors (10). However, the physiological relevance of the AC– AKAP5– GluA1 interaction remained unexplored. This issue is important because on one hand association with AKAP5/PKA inhibits the activity of AC2, AC5, and AC6 (but not AC1, AC8, and AC9) (10) when on the other hand placing ACs in close proximity to PKA-substrate complexes should increase efficacy of the corresponding cAMP-stimulated phosphorylations (12).

AMPA receptors mediate most of the glutamatergic transmission under basal conditions. They are tetramers that are formed by one or more types of the subunits GluA1-4; GluA1/2 heteromers account for the majority of AMPA receptors in the hippocampus with GluA1 homomers and GluA2/3 heteromers contributing a significantly lower portion (13-16). PKA phosphorylates GluA1 on serine 845 (S845) (17,18), which is important for GluA1 surface expression (19-23), activity-induced postsynaptic accumulation (5,24) and various forms of synaptic plasticity (25-29) (but see (30)).

AKAP5 was named AKAP150 in rodents and AKAP79 in humans. AKAP150 is larger than AKAP79 due to an insert of 36 imperfect octapeptide repeats of unknown function (31). The C terminus of AKAP5 (aa 392-416 in AKAP79) anchors the regulatory RII subunits of PKA (32). The N-terminus binds PKC, F-actin, cadherin, and PIP2 and helps to target AKAP150 to dendritic spines (33-37). The central region of AKAP150 binds the Ca2+ and calmodulin-activated phosphatase calcineurin (PP2B) (38-40). This interaction is important for LTD and for curbing LTP (41,42). AKAP5 is the main postsynaptic AKAP (43-47). Functionally, AKAP5 links PKA, PKC, and the antagonistic phosphatase PP2B via SAP97 and perhaps also PSD-95 to GluA1 for

dynamic phosphorylation and dephosphorylation (17,37,48-50). On a molecular level, PKA is physically connected to GluA1 by AKAP5. AKAP5 can bind to the SH3 and GK domains of the postsynaptic scaffolding proteins PSD-95 and SAP97 (48,51) (Fig. 1). PSD-95 interacts with its first and second PDZ domain with stargazin (γ2) and its homologues γ3, γ4, and γ8 (collectively called TARPs), which in turn associate with AMPA receptors for their postsynaptic targeting (52-55). SAP97 can directly bind with its first or second PDZ domain to the C-terminus of GluA1 (56-58) (Fig. 1).

As AKAP5 can recruit AC activity to GluA1 (10), we hypothesized that anchoring of AC by AKAP5 is important for postsynaptic Gs-protein coupled receptor (GsPCR) – Gs – AC – PKA – GluA1 signaling. We systematically compared β adrenergic regulation of S845 phosphorylation and of postsynaptic glutamate receptor responses in AKAP150 KO mice (59) and mice in which the last 36 residues of AKAP150 had been deleted (D36 mice) (43,45-47) to test the functional roles of AKAP150 with respect to PKA vs AC targeting. We find that basal S845 phosphorylation and its upregulation by β adrenergic stimulation is much more drastically impaired in AKAP5 KO than D36 mice. The increase in basal glutamatergic synaptic transmission upon β-adrenergic stimulation is compromised in forebrain slices from AKAP5 D36 and KO mice. However, LTP induced by a 5 Hz/180 sec tetanus, which requires β-adrenergic stimulation in addition to the electric stimulus train, is only modestly reduced in D36 but completely abrogated in KO mice. We conclude that anchoring of AC by AKAP5 is important for positive regulation of postsynaptic functions that include AMPA receptor activity by cAMP-PKA signaling.

EXPERIMENTAL PROCEDURES Reagents and antibodies.(-) Isoproterenol bitartrate salt, ICI118551, CGP20712, microcystin LR, and (+/-) propranolol hydrochloride were from Sigma. IEM1460 was from Tocris. Antibodies against the β1AR (V-19; Lot K1209) and β2AR (H-20; Lot J0305) were from Santa Cruz. The antibodies against synaptophysin, PSD-95, GluA2, GluN1, GluN2B, and ACs (pan-specific) were as given earlier (3,5,56,60-62). The rabbit anti-AC5/6 antibody was from Santa Cruz (C17; sc-590). The phospho-specific antibodies

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against S831 and S845 were produced against the synthetic peptides LIPQQ(pS)INEAIK (GluA1 residues 826-836) and TLPRN(pS)GAGASK (GluA1 residues 840-850; pS: phospho-serine) (see (18)) and the anti-GluA1 antibody against the peptide MSHSSGMPLGATGL, which corresponds to the very C-terminus of GluA1 (residues 894-907). All peptides had been coupled to bovine serum albumin for immunization of rabbits, as described earlier (63). Non-specific control antibodies were from Zymed. Horseradish peroxidase-coupled (HRP) protein A was from Amersham Biosciences. ECL and ECL plus reagents were from GE healthcare. Other reagents were from the typical suppliers and of the usual quality.

Animal use and origin. All procedures followed NIH guidelines and had been approved by the Institutional Animal Care and Use Committees at the University of Iowa and UC Davis. The production of AKAP150 KO mice (by insertion of a neomycin phosphotransferase (neo) cassette into the intron-less AKAP150 gene) and of D36 mice (by creation of a premature stop codon) and there genotyping were described earlier (43,46). Both KO and D36 mouse lines used in the current work had been backcrossed to C57Bl/6 (Taconic Farms) for at least 10 generations. All mice were between 8 and 16 weeks old except for PSD preparations for which up to 8 months old mice were used.

Immunoprecipitation (IP) and immunoblotting. Forebrain slices containing hippocampus (see below) were extracted with a tenfold excess (volume/weight) of Buffer A (150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM Tris-HCl, pH 7.4, and protease inhibitors) containing 1% Triton X-100 with a glass-teflon homogenizer. Samples were cleared from non-solubilized material by ultracentrifugation (250,000 x g for 30 min) before IP with anti-GluA1 (2 µl of antiserum), H20 against the β2AR (8 μg), anti-GluN1 (1 μg), anti-GluN2B (1 μg) or an equivalent amount of non-specific rabbit IgG (typically 2-8 μg; overnight at 4oC) and subsequent immunoblotting as described (60,61). Proteins were separated by SDS-PAGE, transferred over night onto polyvinyldifluoride (PVDF) membranes, incubated with primary antibodies for 1 h, washed, incubated with HRP-protein A for 1 h, and washed for 4 h before detecting ECL or ECL plus signals by film. Multiple exposures with increasing time periods

were obtained to ensure that signals were in the linear range, as described (64,65).

Post-synaptic density (PSD) preparation. For each preparation 4 forebrains per genotype were homogenized on ice with a relatively loosely fitting glass-teflon homogenizer in 10 ml of freshly made ice-cold buffer B (0.32 M sucrose, 1 mM Tris pH 7.4, 1 mM MgCl2) containing the protease inhibitors leupeptin (10 μg/mL), aprotinin (10 μg/mL), pepstatin A (1 μM), and phenylmethylsulfonylfluoride (PMSF; 200 μM), and the phosphatase inhibitor microcystin-LR (2 μM). The lysates were centrifuged at 1,400 x g for 10 min at 4oC. The pellets were rehomogenized in equal volume of buffer B followed by centrifugation (10 min, 710 x g, 4oC). The combined supernatants (Lys) were centrifuged first at 710 x g (10 min) and then at 13,800 x g (10 min) to obtain the P2 fraction enriched with synaptic membranes. P2 was resuspended in 3 ml of buffer C (buffer B without MgCl2), and layered on top of a 0.85/1/1.25M sucrose gradient. After centrifugation at 82,500 x g (2 hrs), the synaptosome enriched interface (Syn) was collected between the 1 M and 1.25 M sucrose layers and extracted with an equal volume of Triton X-100 buffer (1% Triton X-100, 12 mM Tris pH 8.0, 125 mM KCl, plus protease and phosphatase inhibitors as before) for 15 min on ice. The Triton X-100-insoluble material, which is enriched for PSD, was spun down (30 min, 35,000 x g) and resuspended in 1 ml buffer C followed by layering on top of a 1/1.5/2M sucrose gradient. After centrifugation at 225,000 x g (2 h), the PSD-enriched fraction (PSD) was collected from the 1.5/2M sucrose interface. Protein was quantified with a microplate bicinchoninic acid (BCA) assay. 10 μg protein of each fraction were separated by SDS-PAGE and analyzed by immunoblotting.

Preparation and treatment of brain slices. Mice that were typically 8-16 weeks old were decapitated and brains placed into ice-cold artificial cerebrospinal fluid (ACSF, containing in mM: 127 NaCl, 26 NaHCO2, 1.2 KH2PO4, 1.9 KCl, 2.2 CaCl2, 1 MgSO4 and 10 D-glucose, 290-300mOsm/kg). ACSF was saturated with 95% of O2 and 5% of CO2 (final pH 7.3). About 1/3 of the rostral and caudal ends of the brain were trimmed off. 350 µm thick forebrain slices containing hippocampus were prepared with a vibratome (Leica VT 1000A). Slices were kept in oxygenated ACSF for 1 h at 30°C and for 1-5 h at 24°C before used for experiments.

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For phospho-analysis, slices were equilibrated at 32 °C for 30 min and treated with vehicle, ISO (10 μM), or ISO plus propranolol (1 μM) for 5 min. Slices were extracted with 1% deoxycholate before IP with anti-GluA1 and anti-GluN2B and immunoblotting with phospho-specific antibodies against S831 and S845 on GluA1, S897 on GluN1, and finally anti-GluA1 and anti-GluN1 for total GluA1 and GluN1 levels, respectively.

Field EPSP recording. Forebrain slices containing hippocampus were prepared as above and placed in a recording chamber and perfused with ACSF saturated with 95% O2 and 5% CO2 (2 ml/min) at 30oC. Field excitatory postsynaptic potentials (fEPSPs) were evoked in the hippocampal CA1 area by stimulating the Schaffer collateral pathway with a bipolar tungsten electrode every 15 sec. The stimulus intensity was adjusted to induce 50% of the maximal response. The fEPSP was recorded with a glass electrode filled with ACSF. Signals were amplified by an Axopatch 2B amplifier, digitized by a Digidata 1320A and recorded by Clampex 10 (Molecular Devices, CA). The tetanic stimulation was a train of pulses given at the frequency of 5 Hz for 3 min. For quantitative comparisons, the averages of fEPSP initial slopes were calculated from the 5 min immediately preceding the onset of perfusion with ISO, the 5 min immediately before the onset of the tetanus, and the 30 min starting 15 min after the tetanus.

Data analysis. Immunoblot signals were quantified using Photoshop or ImageJ (NIH). All data were analyzed by Excel (Microsoft Corp.) and Prism 4.0 (GraphPad Software, Inc.). Data were shown as mean ± SEM. “n” indicates number of independent experiments. Student’s t test was used for two groups comparison and one- or two-way analysis of variance (ANOVA) was used for comparisons of more than two groups. P < 0.05 was considered statistically significant. RESULTS

AKAP5 is necessary for AC - GluA1 interaction. As AKAP5 interacts with not only PKA but also with at least six AC isoforms (10-12), we hypothesized that the previously described association of GluA1-containing AMPA receptors with AC (5) is mediated by AKAP5, which in turn is linked to GluA1 via SAP97 and possibly PSD-95 (Fig. 1). In fact, the AC-GluA1 co-IP was fully abrogated in AKAP5 KO but not affected in D36 mice as revealed by a pan-specific AC antibody

that recognizes all isoforms, which migrate all with an apparent molecular mass of about 150 kDa (Fig. 2A,B). Given the complete loss of the co-IP of ACs with GluA1 we conclude that AKAP5 is the main and most likely only adaptor protein that links ACs to GluA1. As expected, co-IP of the PKA regulatory RIIα subunit with GluA1 was strongly reduced in both genotypes (Fig. 2A,B). The incomplete loss of PKA association with GluA1 in D36 mice could be due to additional AC interactions with other AKAPs within the overall complex. In agreement with this notion is the nearly complete loss of PKA in parallel with AC in the AKAP5 KO mice. Total amounts of AC or RIIα were unaltered in brain lysates (Fig. 2 C,D). AC5 and AC6 interact with the second of the three poly-basic regions in the N-terminus of AKAP5 (10). We found that an AC5/6-selective antibody recognized the appropriate AC bands in IPs of GluA1 but not GluN1 (Fig. 2E,F). Accordingly, AC5/6 is associated with GluA1-containing AMPA receptors but not NMDA receptors.

Anchoring of both, PKA and AC, by AKAP5 is important for S845 phosphorylation. AKAP5 functionally and structurally links PKA to GluA1 for S845 phosphorylation, an important regulatory mechanism for GluA1 (48,49,50). Given our finding that AKAP5 also recruits ACs to GluA1, we evaluated whether S845 phosphorylation is more severely affected in AKAP5 KO than D36 mice as a result of the loss of AC anchoring in addition to the loss of PKA anchoring in KO mice. In fact, basal phosphorylation levels of S845 were unaltered in whole forebrain slices of D36 mice but strongly reduced in KO (Fig. 3A, upper panels, 3C, left graph). At most 10%, perhaps even much fewer GluA1 subunits, are phosphorylated on S845 under basal conditions (22,66). However, the β adrenergic agonist isoproterenol (ISO) stimulates S845 phosphorylation several fold (5,6,29,67). The ISO-stimulated increase in S845 phosphorylation was more than 5 fold for WT slices but less than 3 fold for KO and D36 slices (Fig. 3A, upper panels, 3D, left graph). This reduction in efficacy in upregulation of S845 phosphorylation, paired with the much lower level of basal phosphorylation in KO mice, translates into a dramatically reduced total ISO-induced phosphorylation in KO mice compared to WT. Although the fold increase in S845

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phosphorylation by ISO is also strongly impaired in D36 vs WT mice, given that D36 mice had near WT levels of basal S845 phosphorylation, the loss in total ISO-stimulated phosphorylation is much more modest in D36 than KO mice. Augmentation of S845 phosphorylation by ISO was blocked by the general β-adrenergic antagonist propranolol confirming that ISO-induced S845 phosphorylation was mediated by β-adrenergic signaling.

Basal phosphorylation levels for S831, a PKC and CaMKII site (17,18), were not statistically different in D36 and KO vs. WT slices indicating that loss of PKC anchoring to GluA1 by AKAP5 in the KO mice had no significant effect on basal S831 phosphorylation (Fig. 3A, middle panels, 3D, left graph). Also, ISO did not significantly stimulate S831 phosphorylation. This result was expected because PKC is typically activated by Gq- and not Gs-coupled receptors and there is no evidence that either PKC or CaMKII would be activated upon stimulation of the β AR - AC - cAMP - PKA cascade at postsynaptic sites. We also monitored phosphorylation of S897 in the C-terminus of the NMDA-type glutamate receptor GluN1 subunit, which is an established PKA site of unknown function (68). Neither basal S897 phosphorylation levels nor ISO-induced S897 phosphorylation were affected in AKAP5 D36 or KO mice (Fig. 3B, upper panels, 3C,D, right graphs). Accordingly, loss of AKAP5 function affects AMPA receptor but not NMDA receptor phosphorylation by PKA. The NMDA receptor can bind PKA and AC1, AC2, AC3, and AC9 via the AKAP yotiao (9,69), which might thus functionally be the more important if not the only PKA and AC anchor for NMDA receptors. Our results indicate that the deficit in GluA1 S845 phosphorylation in AKAP5 D36 and KO mice is not a universal deficit in PKA-mediated phosphorylation in dendritic spines.

AKAP5 is not necessary for β2 AR - GluA1 interaction. The β2 AR is linked to GluA1 via PSD-95 and stargazin and their homologues (5). The β2 AR also binds directly to AKAP5 (70,71). More precisely, the β2 AR C-terminus interacts with the N-terminal ~200 residues of AKAP5 (71). As AKAP5 binds to PSD-95 and SAP-97 on one hand and the β2 AR on the other, it is conceivable that AKAP5 is required for or contributes to stabilization of the β2 AR - PSD-95 – stargazin - GluA1 complex in addition to the direct interactions of the β2 AR with PSD-95 and SAP97. We thus tested whether impaired ISO-

induced S845 phosphorylation in AKAP5 mutant mice could be due to loss of β2 AR from the GluA1 complex in addition to loss of AC. However, co-IP of GluA1 as well as GluA2, the other main AMPA receptor subunit in forebrain, with the β2 AR was comparable for WT, D36 and KO mice (Fig 4A,B). Accordingly, loss of AKAP5 does not affect the formation or stability of the β2 AR-GluA1 complex.

To ensure specificity of the β2 AR antibody, we IPed both, the β1 AR and the β2 AR from WT mouse brain. Subsequent immunoblotting with either antibody shows β1 AR immunoreactivity in the β1 AR but not β2 AR IP and β2 AR immunoreactivity in the β2 AR but not β1 AR IP at the correct MR of ~ 60 kD in both cases (Fig. 4C). Accordingly, either antibody recognizes its cognate target protein without cross-reacting with the other isoform.

To scrutinize whether the coIP of GluA1, AKAP5 (Fig. 4A,B) and of AC5/6 (Fig. 2E,F) with the β2 AR are truly due to IP of the latter and not an off target cross-reacting protein, we IPed the β2 AR from WT and β2 AR KO mouse brains. AC5/6, AKAP5, and GluA1 coIPed only from WT but not from β2 AR KO mice (Fig. 4D). We conclude that these coIPs are due to IP of the β2 AR indicating association of these proteins with the latter.

AKAP5 is required for postsynaptic targeting of AC in general and specifically of AC5/6. To test whether AKAP5 is important for postsynaptic localization of ACs, we isolated PSD fractions from brains of WT, AKAP5 KO, and D36 mice for immunoblot analysis. The purity of the final PSD fractions was reflected by the loss of synaptophysin signal and the strong enrichment of the PSD marker PSD-95 (Fig. 5A,C). Immunoreactivity of our pan-specific antibody was drastically reduced in AKAP5 KO but not D36 mice (Fig. 5A-D).

Because AKAP5 specifically links AC5/6 to GluA1, we analyzed AC5/6 immunoreactivity in PSD preparations from AKAP5 KO mice. As for panAC signals, the AC5/6 signals were dramatically decreased in the KO mice (Fig. 5A-D). These findings indicate that AKAP5 is the most critical docking protein for postsynaptic targeting of ACs and especially AC5/6, which is analogous to its central role in postsynaptic localization of PKA (43).

AKAP5 is not necessary for postsynaptic targeting of the β2 AR. AKAP5 could more generally link the β2 AR to the PSD. To control

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for this possibility, we analyzed the β2 AR content of the PSD fractions from WT, D36 and KO mice by immunoblotting. The β2 AR was enriched in parallel with PSD-95 and GluA1 (Fig. 5A,C) illustrating for the first time by such subcellular fractionation that the β2 AR is a component of the PSD. There was no difference between WT, D36 and KO brains with respect to the β2 AR content of PSDs (Fig. 5B,D) demonstrating that neither the D36 deletion nor the complete KO affects postsynaptic targeting of the β2 AR. Collectively these results indicate that deficits in ISO-induced S845 phosphorylation are not due to mistargeting of the β2 AR away from the PSD in general and specifically from GluA1.

We also monitored subcellular distribution of the β1 AR. We observed only in pre-PSD fractions β1 AR immunoreactivity but not in the PSD fractions themselves (Fig. 5A,C). Accordingly, the β1 AR is not enriched, perhaps even absent in PSDs in agreement with our recent findings that the β1 AR contributes much less than the β2 AR to postsynaptic signaling if at all (29).

AKAP5 is important for S845 phosphorylation in the hippocampus. For a physiological evaluation of the relevance of AC anchoring by AKAP5 we turned our attention to the hippocampus. ISO treatment increased GluA1 S845 phosphorylation in acute hippocampal slices by 60% (Fig. 6A, upper panel, 6B), which is a much smaller increase than in whole forebrain slices. This much more modest response is in agreement with our earlier observation that ISO increased S845 phosphorylation in hippocampal cultures by 80% (5). It is also consistent with our previous observation that ISO affects basal synaptic transmission much more severely and robustly in the prefrontal cortex than in the hippocampal CA1 area (5). Furthermore, hippocampal samples did not show a decrease in basal S845 phosphorylation in contrast to the forebrain sections (compare Fig. 3C, left graph, with Fig. 6A, upper panel, 6B) possibly because basal β adrenergic tone might be lower at postsynaptic sites in the hippocampus than in the cortex. As in the forebrain samples, GluA1 S831 phosphorylation was not affected in AKAP5 KO mice nor did ISO increase it in the hippocampus (Fig. 6A,C).

Upregulation of postsynaptic AMPA receptor responses by β adrenergic signaling requires AKAP5. LTP induced by prolonged theta rhythm tetani (5 Hz for 180 s; prolonged theta-tetanus-LTP or PTT-LTP) at Schaffer collateral – CA1

synapses is an important form of synaptic plasticity because the theta-rhythm (5-12Hz) is a prominent activity pattern of the hippocampus (72,73). The dependence of PTT-LTP on β adrenergic stimulation of adenylyl cyclase and PKA (27,29,74-76) is fundamentally different from more standard LTP triggered by multiple tetani of 50-100 Hz or theta-burst stimulations, which does not require PKA at all or, if induced by a single tetanus, only requires basal PKA activity (43). PTT-LTP is impaired in phosphorylation-deficient GluA1 S831A/S845A double KI mice (27) and in GluA1 S845A single KI mice (29). It does not require β adrenergic stimulation in phosphorylation-mimetic GluA1 S831D/S845D double KI mice (77). Accordingly, S845 phosphorylation is not only necessary but also sufficient to gate induction of PTT-LTP that would otherwise require β adrenergic stimulation for gating. Given the simultaneous loss of AC and PKA from GluA1 in AKAP5 KO mice vs. the loss of only PKA in D36 mice we hypothesized that upregulation of postsynaptic responses and PTT-LTP are more severely affected in KO than D36 mice.

Similar to our previous findings (5), in acute slices from WT mice ISO by itself increased fEPSP initial slopes in only ~60% of the recordings with little to no effect in the remaining ~40% (Fig. 7A,B). D36 and KO mice never showed any increase in the postsynaptic response upon perfusion with ISO suggesting that the ISO effect observed under basal conditions specifically requires anchoring of PKA by AKAP5 (Fig. 7C-E).

In WT slices induction of PTT-LTP increased fEPSPs by 67% if ISO by itself had initially no effect (Fig. 7A,E) and by 30% otherwise (Fig. 7B,E). The reduced degree of potentiation in slices that showed an increase to the ISO perfusion suggests that this increase might occlude a portion of PTT-LTP. Accordingly, the two regulatory mechanisms may share molecular mechanisms. Importantly, induction of PTT-LTP increased the fEPSP response by only 33% in D36 mice (Fig. 7C,E). This finding indicates that PTT-LTP is significantly lower in D36 slices when compared to responses in WT slices in which the basal response to ISO was absent as was the case in D36 slices. Most relevant with respect to the role of AKAP5 in AC targeting is the observation that KO mice did not show any PTT-LTP at all (Fig. 7D,E). These results indicate that AKAP5-

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mediated anchoring of not only PKA but also specifically ACs is critical for PTT-LTP.

Upregulation of basal synaptic transmission is mediated by β2 AR. We recently demonstrated that PTT-LTP requires the β2 AR but not the β1 AR (29). Given that some recordings showed an increase in basal transmission upon ISO application whereas others did not, we tested whether the difference could be due to differential β1 vs β2 AR contribution. Although the β1 AR appears to be absent from the PSD (Fig. 5A) it could be present and regulate AMPA receptor availability in the perisynaptic space surrounding the PSD. However, the increase in basal response by ISO was completely blocked by 40 nM of the β2 AR-specific antagonist ICI118551 (Fig. 8A,C) but not by 1 μM the β1 AR-specific antagonist CGP20712 (Fig. 8B,C). Importantly, we recently confirmed that the CGP20127 batch we used in the current experiments is active (29). The findings that ICI118551 completely blocked the basal ISO effect whereas CGP20712 had no effect collectively demonstrate that the basal ISO effect is mediated by the β2 but not β1 AR.

PPT-LTP requires activity of GluA2-lacking AMPA receptors. Despite the fact that S845 phosphorylation was dramatically reduced in our KO vs WT mice, basal synaptic transmission was normal in 8-12 week old KO and also D36 mice (47). Although S845 phosphorylation upregulates GluA1 surface expression upon stimulation of PKA (19-23), S845 phosphorylation does not seem to be an important determinant for postsynaptic responses under basal conditions as phosphorylation-deficient S845A KI mice have normal basal synaptic transmission (30). However, S845 phosphorylation is important for activity-driven postsynaptic accumulation of homomeric GluA1 receptors that are formed by ectopic expression of GluA1 (24). Notably such GluA2-lacking AMPA receptors are usually not contributing to excitatory postsynaptic currents (EPSCs) of CA1 pyramidal cells under basal conditions (15,78) but become apparent upon certain forms of synaptic potentiation (79-83) (but see (78)). Contrasting GluA2-containing AMPARs, these GluA1 homomers have a higher single channel conductance and are Ca2+ permeable and can, therefore, play an important signaling role under certain conditions (84). Although GluA3 and GluA4 subunits can also form GluA2-lacking, Ca2+ permeable receptors, the prevailing GluA2-lacking receptors in CA1 are likely GluA1 homomers (14,15).

Because GluA1 homomeric receptors have four S845 residues for PKA phosphorylation sites rather than two as in GluA1/A2 heteromers, the main AMPA receptor species in CA1 (15), it is possible that S845 phosphorylation has a stronger effect on the homomers than heteromers with respect to their activity-induced postsynaptic targeting. In this way S845 phosphorylation could help to drive those GluA2-lacking GluA1 homomers under certain forms of synaptic plasticity to postsynaptic sites. Given the congruence in dependency of PTT-LTP on S845 phosphorylation (29) and on AC anchoring by AKAP5 (Fig. 7); given that PTT-LTP strictly depends on activation of AC (and PKA) by β-adrenergic receptors (74); and given that β adrenergically induced S845 phosphorylation is heavily blunted in AKAP5 KO mice (Fig. 3A, upper panel, 3C and D left graphs; Fig. 6A, upper panel, B) we hypothesized that PTT-LTP depends on GluA1 phosphorylation on S845 because it requires at least temporarily GluA2-lacking AMPA receptors. Such finding would not only provide further support for our hypothesis that AC anchoring by AKAP5 is important for S845 phosphorylation at postsynaptic sites and thereby for PTT-LTP but also expand our knowledge of molecular details underlying PTT-LTP. We inhibited Ca2+ permeable, GluA2-lacking AMPA receptors with IEM1460, which has higher selectivity for those receptors than alternative inhibitors such as philanthotoxin-433 (85-87). IEM1460 completely blocked PTT-LTP (Fig. 8A,D). Because IEM1460 can also inhibit at higher concentrations NMDA receptors and those receptors are thought to contribute to PTT-LTP (74), we tested in parallel whether IEM1460 affected LTP induced by two tetani of 100 Hz/1 sec, which depends on NMDA receptors but not on GluA2-lacking AMPAR in 8-12 week old mice (43,86). IEM1460 had no effect at all on this 2x100Hz/1 sec LTP (Fig. 7B-D) indicating that it did not impair NMDA receptor function in postsynaptic signaling and synaptic plasticity. Accordingly, IEM1460 prevented PTT-LTP by acting on GluA2-lacking AMPA receptors rather than NMDA receptors under our conditions.

DISCUSSION

Importance of AC anchoring by AKAP5 in postsynaptic signaling. Interaction of various ACs

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with GluA1 has recently been observed (5,10) but the functional relevance of this interaction had so far not been evaluated. Association with the AKAP5-PKA complex inhibits the activity of some (AC2, AC5, and AC6) but not other AC isoforms (AC1, AC8, and AC9) (10). Inhibition of AC2, AC5, and AC6 is likely mediated at least in part by negative feedback by PKA on the activity of those ACs (7). Yet localization of ACs in close vicinity to PKA-substrate assemblies should augment signaling from AC to PKA via cAMP (12). Whether the interaction of ACs with GluA1 via AKAP5 increases or decreases the AC-PKA signaling in this complex was thus not predictable, although conceptually the former appeared more likely in our view, at least during the initial phase of AC activation. By comparing the effects of AKAP5 D36 deletion with full AKAP5 KO we find that the loss of PKA plus AC anchoring in the KO mice impairs phosphorylation of GluA1 on S845 and postsynaptic regulation of AMPA receptor activity more severely than loss of PKA anchoring alone in D36 mice. We conclude that AKAP5-mediated docking of both, AC and PKA, to GluA1 is important for optimal signaling from AC to PKA and ultimately GluA1. Regulation of NMDA receptor phosphorylation on GluN1 S897 by PKA is not affected in either genotype indicating the importance of highly localized targeting and thereby signaling of both, AC and PKA within dendritic spines.

As yotiao has the potential to recruit PKA and AC to NMDA receptors, the nearly complete loss of panAC immunoreactivity in PSDs from AKAP5 KO mice is puzzling (Fig. 5A,B). The same is true for the dramatic loss of PKA from PSDs in D36 mice (43) and might reflect that either yotiao along with the associated AC and PKA is easier extracted from PSDs than AKAP5 or that the interactions of AC and PKA with yotiao are easier disrupted by Triton X-100, which is required to remove presynaptic elements from the PSDs.

Relevance of basal S845 phosphorylation. S845 phosphorylation is unaltered under basal conditions in D36 mice but dramatically reduced in forebrain of KO vs WT mice. A different AKAP5 KO shows a similar reduction in basal S845 phosphorylation (44). At the same time basal synaptic transmission is normal in 8-16 week old KO mice (47) as it is in D36 mice (43). We only analyzed mice of such an age here because synaptic transmission is actually increased in younger AKAP5 KO and D36 mice

due to increases in synapse density; however, this deficit is rectified during postnatal development so that synapse density becomes normal in the young adult KO and D36 mice (47). Given that S845 phosphorylation increases surface expression of GluA1 (19-23) and this phosphorylation is strongly reduced in AKAP5 KO mice (Fig. 3C), the fact that basal synaptic transmission is normal in the AKAP5 KO mice under basal conditions appears at the first glance surprising. However, at most 10% but perhaps a much smaller percentage of GluA1 subunits are phosphorylated on S845 under basal conditions (22,66). It is thus possible that this basally low level of phosphorylation has a minimal effect on postsynaptic targeting and activity of AMPA receptors. Phosphorylation-deficient S845A KI mice have normal basal synaptic transmission indicating that S845 phosphorylation is indeed not important for regulating AMPA receptor responses under basal conditions (30).

Differential effect of AKAP5 D36 vs KO on 100 Hz LTP vs PTT-LTP. In contrast to WT mice, LTP induced by a single 100 Hz/1 sec tetanus is nearly absent in adult D36 mice and LTD is impaired in 12-14 day old D36 mice (43,45,46). Remarkably, adult AKAP5 KO mice had normal 100 Hz LTP and P12-14 KO mice normal LTD (but see (44) for deficits in adult LTD (but not LTP) in another AKAP5 KO mouse) and thus a milder phenotype than the D36 mutation (46). One potential explanation for the fact that D36 but not KO mice have a severe 100 Hz LTP deficit is that in the KO but not D36 mice another AKAP could fill in for AKAP5 in certain complexes, possibly one that recruits only PKA but not PP2B causing a shift towards higher phosphorylation. Although there is currently no evidence for such compensation (46,59), it is difficult to rule this possibility out. In fact, AKAP12 (gravin, AKAP250) has recently been shown to be important for PKA-dependent forms of LTP induced by 100 Hz tetani and thus is a potential candidate that could at least partially compensate for loss of AKAP5 from postsynaptic sites (88). Interestingly, this work shows that AKAP12 is also important for PTT-LTP although loss of AKAP12 does not affect phosphorylation of GluA1 on S845 (88). Rather the function of AKAP12 in synaptic plasticity (88) appears to be mainly to recruit PKA to the β2 AR to foster its phosphorylation by PKA on S345 and S346 (89-91), which switches the coupling of the β2 AR from Gs/cAMP/PKA to Gi/ERK (92,93).

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Another potential explanation for the finding that D36 but not AKAP5 KO mice are strongly impaired in 100 Hz LTP is that the full KO of AKAP150 eliminated not only PKA but also PP2B anchoring, thereby less severely shifting phosphorylation of certain targets towards dynamic dephosphorylation than the D36 mutation, which preserves PP2B anchoring (46). However, AKAP5 KO but not D36 mice show a strong impairment in S845 phosphorylation. Accordingly, it appears that targets other than S845 must be more strongly affected in D36 vs. AKAP5 KO mice at least under the basal conditions and their phosphorylations must be important for 100Hz LTP. Alternative targets that are present at postsynaptic sites or dendrites and require AKAP5-anchored PKA for their regulation are the L-type Ca2+ channel Cav1.2 (3,40,59,94,95) and the K+ channel Kv4.2 (96). Phosphorylation of Kv4.2 on S552 by PKA fosters internalization of Kv4.2, which in turn increases neuronal excitability (96,97), thereby potentially fostering 100 Hz LTP. If Kv4.2 phosphorylation is more substantially decreased in D36 than KO mice, it would augment surface expression of Kv4.2, thereby reducing excitability and making it more difficult to induce LTP. L-type Ca2+ channels contribute to LTP induced by 200 Hz tetani (98,99). Cav1.2 channel activity is increased by PKA (71). The reduction in phosphorylation of Cav1.2 by PKA that is observed in D36 mice (59) could thus potentially affect LTP induction by a single 100 Hz tetanus although L-type channels are typically not required for LTP induced by several 100 Hz tetani.

The requirement of PTT-LTP for AKAP5 shown here and for S845 phosphorylation described recently (29) clearly differs from the corresponding requirements of 100Hz LTP. Adult SS831/845AA double KI mice are deficient in LTP induced by bursts of 100 Hz (thetaburst-LTP) (25), which is similar to regular 100 Hz LTP. However, neither S831A nor S845A single KI mice show this loss in thetaburst-LTP (30), which is analogous to the fact that single 100 Hz LTP is normal in AKAP5 KO mice (46). Accordingly, neither S831 nor S845 phosphorylation is strictly required for LTP induced by high frequency

stimuli as long as one or the other site is available. In other words, the presence of S831 as PKC and CaMKII target site and S845 as PKA target site safe guards against impaired phosphorylation of one of the two sites, allowing for 100Hz LTP even if one of the two sites is unavailable as in S831A and S845A single KI mice (30) or if PKA anchoring by AKAP5 for effective S845 phosphorylation is abrogated (46). In contrast, PTT-LTP requires S845 phosphorylation (29). Accordingly, S831 phosphorylation cannot substitute for loss of S845 phosphorylation in PTT-LTP explaining the complete absence of PTT-LTP in AKAP5 KO mice in which S845 phosphorylation is more severely affected than in D36 mice. This finding also indicates that S831 and S845 are not equivalent although molecular differences in their mechanistic functions might be modest.

AKAP5 anchors not only PKA but also PKC. It is conceivable that loss of PKC anchoring in the KO contributes to the complete abrogation of PTT-LTP whereas the D36 mutant still can provide PKC targeting, which might be sufficient for a partial PTT-LTP. However, basal S831 phosphorylation was not altered in AKAP5 KO mice, β adrenergic stimulation did not lead to a significant increase in this phosphorylation, and S845A KI mice show little if any PTT-LTP (29), making a major contribution of S831 phosphorylation by AKAP5-anchored PKC to PTT-LTP unlikely. Nevertheless, these observations do not exclude that PKC targets other than S831 are involved in PTT-LTP.

Potential role of AC anchoring by AKAP5 in vivo. Norepinephrine fosters arousal and learning especially under novel and emotionally charged situations via β adrenergic signaling (27,100-106). β adrenergic signaling facilitates several forms of LTP in the hippocampal dentate gyrus and CA1 region of the hippocampus (27,29,74-76,107,108). Thus our findings demonstrating that anchoring of both, PKA and AC, by AKAP5 is important for β adrenergic stimulation of S845, a critical PKA site at postsynaptic sites of glutamatergic synapses, and for PTT-LTP, implicate AC anchoring by AKAP5 as a relevant molecular component in the regulation of alertness by norepinephrine.

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REFERENCES

1. Wong, W., and Scott, J. D. (2004) AKAP signalling complexes: focal points in space and time. Nat. Rev. Mol. Cell. Biol. 5, 959-970

2. Smith, F. D., Langeberg, L. K., and Scott, J. D. (2006) The where's and when's of kinase anchoring. Trends Biochem. Sci. 31, 316-323

3. Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Burette, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) A β2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. [see comments]. [erratum appears in Science 2001 Aug 3;293(5531):804]. Science. 293, 98-101

4. Balijepalli, R. C., Foell, J. D., Hall, D. D., Hell, J. W., and Kamp, T. J. (2006) From the Cover: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for beta2-adrenergic regulation. Proc. Natl. Acad. Sci. U. S. A. 103, 7500-7505

5. Joiner, M. L., Lise, M. F., Yuen, E. Y., Kam, A. Y., Zhang, M., Hall, D. D., Malik, Z. A., Qian, H., Chen, Y., Ulrich, J. D., Burette, A. C., Weinberg, R. J., Law, P. Y., El-Husseini, A., Yan, Z., and Hell, J. W. (2010) Assembly of a β2-adrenergic receptor-GluR1 signalling complex for localized cAMP signalling. EMBO J. 29, 482-495

6. Wang, D., Govindaiah, G., Liu, R., De Arcangelis, V., Cox, C. L., and Xiang, Y. K. (2010) Binding of amyloid beta peptide to β2 adrenergic receptor induces PKA-dependent AMPA receptor hyperactivity. FASEB J. 24, 3511-3521

7. Bauman, A. L., Soughayer, J., Nguyen, B. T., Willoughby, D., Carnegie, G. K., Wong, W., Hoshi, N., Langeberg, L. K., Cooper, D. M., Dessauer, C. W., and Scott, J. D. (2006) Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes. Mol. Cell. 23, 925-931

8. Kapiloff, M. S., Piggott, L. A., Sadana, R., Li, J., Heredia, L. A., Henson, E., Efendiev, R., and Dessauer, C. W. (2009) An adenylyl cyclase-mAKAPbeta signaling complex regulates cAMP levels in cardiac myocytes. J. Biol. Chem. 284, 23540-23546

9. Piggott, L. A., Bauman, A. L., Scott, J. D., and Dessauer, C. W. (2008) The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain. Proc. Natl. Acad. Sci. U. S. A. 105, 13835-13840

10. Efendiev, R., Samelson, B. K., Nguyen, B. T., Phatarpekar, P. V., Baameur, F., Scott, J. D., and Dessauer, C. W. (2010) AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC5 and -6 to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors. J. Biol. Chem. 285, 14450-14458

11. Willoughby, D., Masada, N., Wachten, S., Pagano, M., Halls, M. L., Everett, K. L., Ciruela, A., and Cooper, D. M. (2010) AKAP79/150 interacts with AC8 and regulates Ca2+-dependent cAMP synthesis in pancreatic and neuronal systems. J. Biol. Chem. 285, 20328-20342

12. Nichols, C. B., Rossow, C. F., Navedo, M. F., Westenbroek, R. E., Catterall, W. A., Santana, L. F., and McKnight, G. S. (2010) Sympathetic Stimulation of Adult Cardiomyocytes Requires Association of AKAP5 With a Subpopulation of L-Type Calcium Channels. Circ. Res. 107, 747-756

13. Hollmann, M., and Heinemann, S. (1994) Cloned glutamate receptors. Annu. Rev. Neurosci. 17, 31-108

14. Wenthold, R. J., Petralia, R. S., Blahos, J., II, and Niedzielski, A. S. (1996) Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons. J. Neurosci. 16, 1982-1989.

15. Lu, W., Shi, Y., Jackson, A. C., Bjorgan, K., During, M. J., Sprengel, R., Seeburg, P. H., and Nicoll, R. A. (2009) Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron. 62, 254-268

16. Traynelis, S. F., Wollmuth, L. P., McBain, C. J., Menniti, F. S., Vance, K. M., Ogden, K. K., Hansen, K. B., Yuan, H., Myers, S. J., and Dingledine, R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405-496

at University of C

alifornia, Davis, on M

ay 8, 2013w

ww

.jbc.orgD

ownloaded from

Role of AC Anchoring by AKAP5 in Postsynaptic Signaling

11

17. Roche, K. W., O'Brien, R. J., Mammen, A. L., Bernhardt, J., and Huganir, R. L. (1996) Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron. 16, 1179-1188

18. Mammen, A. L., Kameyama, K., Roche, K. W., and Huganir, R. L. (1997) Phosphorylation of the a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II. J. Biol. Chem. 272, 32528-32533

19. Ehlers, M. D. (2000) Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting. Neuron. 28, 511-525

20. Swayze, R. D., Lise, M. F., Levinson, J. N., Phillips, A., and El-Husseini, A. (2004) Modulation of dopamine mediated phosphorylation of AMPA receptors by PSD-95 and AKAP79/150. Neuropharmacology. 47, 764-778

21. Sun, X., Zhao, Y., and Wolf, M. E. (2005) Dopamine receptor stimulation modulates AMPA receptor synaptic insertion in prefrontal cortex neurons. J. Neurosci. 25, 7342-7351

22. Oh, M. C., Derkach, V. A., Guire, E. S., and Soderling, T. R. (2006) Extrasynaptic Membrane Trafficking Regulated by GluR1 Serine 845 Phosphorylation Primes AMPA Receptors for Long-term Potentiation. J. Biol. Chem. 281, 752-758

23. Man, H.-Y., Sekine-Aizawa, Y., and Huganir, R. (2007) Regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking through PKA phosphorylation of the Glu receptor 1 subunit. Proc. Natl. Acad. Sci. U. S. A. 104, 3579-3584

24. Esteban, J. A., Shi, S. H., Wilson, C., Nuriya, M., Huganir, R. L., and Malinow, R. (2003) PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat. Neurosci. 6, 136-143

25. Lee, H. K., Takamiya, K., Han, J. S., Man, H., Kim, C. H., Rumbaugh, G., Yu, S., Ding, L., He, C., Petralia, R. S., Wenthold, R. J., Gallagher, M., and Huganir, R. L. (2003) Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell. 112, 631-643

26. Seol, G. H., Ziburkus, J., Huang, S., Song, L., Kim, I. T., Takamiya, K., Huganir, R. L., Lee, H. K., and Kirkwood, A. (2007) Neuromodulators control the polarity of spike-timing-dependent synaptic plasticity. Neuron. 55, 919-929

27. Hu, H., Real, E., Takamiya, K., Kang, M. G., Ledoux, J., Huganir, R. L., and Malinow, R. (2007) Emotion Enhances Learning via Norepinephrine Regulation of AMPA-Receptor Trafficking. Cell. 131, 160-173

28. He, K., Song, L., Cummings, L. W., Goldman, J., Huganir, R. L., and Lee, H. K. (2009) Stabilization of Ca2+-permeable AMPA receptors at perisynaptic sites by GluR1-S845 phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 106, 20033-20038

29. Qian, H., Matt., L., Zhang, M., Nguyen, M., Patriarchi, T., Koval, O. N., Anderson, M. E., He, K., Lee, H.-K., and Hell, J. W. (2012) β2 Adrenergic Receptor Supports Prolonged Theta Tetanus - induced LTP. J. Neurophysiol. 107, 2703-2712

30. Lee, H. K., Takamiya, K., He, K., Song, L., and Huganir, R. L. (2010) Specific roles of AMPA receptor subunit GluR1 (GluA1) phosphorylation sites in regulating synaptic plasticity in the CA1 region of hippocampus. J. Neurophysiol. 103, 479-489

31. Rubin, C. S. (1994) A kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim. Biophys. Acta. 1224, 467-479

32. Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Cone, R. D., and Scott, J. D. (1992) Localization of the cAMP-dependent protein kinase to the postsynaptic densitye by A-kinase anchoring proteins. J. Biol.Chem. 267, 16816-16823

33. Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., and Scott, J. D. (1996) Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science. 271, 1589-1592

at University of C

alifornia, Davis, on M

ay 8, 2013w

ww

.jbc.orgD

ownloaded from

Role of AC Anchoring by AKAP5 in Postsynaptic Signaling

12

34. Dell'Acqua, M. L., Faux, M. C., Thorburn, J., Thorburn, A., and Scott, J. D. (1998) Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5- bisphosphate. EMBO J. 17, 2246-2260

35. Gomez, L. L., Alam, S., Smith, K. E., Horne, E., and Dell'Acqua, M. L. (2002) Regulation of A-kinase anchoring protein 79/150-cAMP-dependent protein kinase postsynaptic targeting by NMDA receptor activation of calcineurin and remodeling of dendritic actin. J. Neurosci. 22, 7027-7044

36. Gorski, J. A., Gomez, L. L., Scott, J. D., and Dell'Acqua, M. L. (2005) Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells. Mol. Biol. Cell. 16, 3574-3590

37. Tavalin, S. J. (2008) AKAP79 Selectively Enhances Protein Kinase C Regulation of GluR1 at a Ca2+-Calmodulin-dependent Protein Kinase II/Protein Kinase C Site. J. Biol. Chem. 283, 11445-11452

38. Coghlan, V. M., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science. 267, 108-111

39. Oliveria, S. F., Gomez, L. L., and Dell'Acqua, M. L. (2003) Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy. J. Biol. Chem. 160, 101-112

40. Oliveria, S. F., Dell'acqua, M. L., and Sather, W. A. (2007) AKAP79/150 Anchoring of Calcineurin Controls Neuronal L-Type Ca(2+) Channel Activity and Nuclear Signaling. Neuron. 55, 261-275

41. Jurado, S., Biou, V., and Malenka, R. C. (2010) A calcineurin/AKAP complex is required for NMDA receptor-dependent long-term depression. Nat. Neurosci. 13, 1053-1055

42. Sanderson, J. L., Gorski, J. A., Gibson, E. S., Lam, P., Freund, R. K., Chick, W. S., and Dell'Acqua, M. L. (2012) AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors. J. Neurosci. 32, 15036-15052

43. Lu, Y., Allen, M., Halt, A. R., Weisenhaus, M., Dallapiazza, R. F., Hall, D. D., Usachev, Y. M., McKnight, G. S., and Hell, J. W. (2007) Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP. EMBO J. 26, 4879-4890

44. Tunquist, B. J., Hoshi, N., Guire, E. S., Zhang, F., Mullendorff, K., Langeberg, L. K., Raber, J., and Scott, J. D. (2008) Loss of AKAP150 perturbs distinct neuronal processes in mice. Proc. Natl. Acad. Sci. U. S. A. 105, 12557-12562

45. Lu, Y., Zhang, M., Lim, I. A., Hall, D. D., Allen, M. L., Medvedeva, Y., McKnight, G. S., Usachev, Y. M., and Hell, J. W. (2008) AKAP150-anchored PKA activity is important for LTD during its induction phase. J. Physiol. 586, 4155-4164

46. Weisenhaus, M., Allen, M. L., Yang, L., Lu, Y., Nichols, C. B., Su, T., Hell, J. W., and McKnight, G. S. (2010) Mutations in AKAP5 disrupt dendritic signaling complexes and lead to electrophysiological and behavioral phenotypes in mice. PLoS One. 5, e10325

47. Lu, Y., Zha, X. M., Kim, E. Y., Schachtele, S., Dailey, M. E., Hall, D. D., Strack, S., Green, S. H., Hoffman, D. A., and Hell, J. W. (2011) A kinase anchor protein150 (AKAP150)-associated protein kinase A limits dendritic spine density. J. Biol. Chem. 286, 26496-26506

48. Colledge, M., Dean, R. A., Scott, G. K., Langeberg, L. K., Huganir, R. L., and Scott, J. D. (2000) Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron. 27, 107-119

49. Tavalin, S. J., Colledge, M., Hell, J. W., Langeberg, L. K., Huganir, R. L., and Scott, J. D. (2002) Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression. J. Neurosci. 22, 3044-3051

50. Hoshi, N., Langeberg, L. K., and Scott, J. D. (2005) Distinct enzyme combinations in AKAP signalling complexes permit functional diversity. Nat. Cell. Biol. 7, 1066-1073

51. Bhattacharyya, S., Biou, V., Xu, W., Schluter, O., and Malenka, R. C. (2009) A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptors. Nat. Neurosci. 12, 172-181

at University of C

alifornia, Davis, on M

ay 8, 2013w

ww

.jbc.orgD

ownloaded from

Role of AC Anchoring by AKAP5 in Postsynaptic Signaling

13

52. Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N. T., Kawasaki, Y., Wenthold, R. J., Bredt, D. S., and Nicoll, R. A. (2000) Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature. 408, 936-943.

53. El-Husseini, A. E., Schnell, E., Chetkovich, D. M., Nicoll, R. A., and Bredt, D. S. (2000) PSD-95 involvement in maturation of excitatory synapses. Science. 290, 1364-1368

54. Schnell, E., Sizemore, M., Karimzadegan, S., Chen, L., Bredt, D. S., and Nicoll, R. A. (2002) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc. Natl. Acad. Sci. U. S. A. 99, 13902-13907

55. Jackson, A. C., and Nicoll, R. A. (2011) The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron. 70, 178-199

56. Leonard, A. S., Davare, M. A., Horne, M. C., Garner, C. C., and Hell, J. W. (1998) SAP97 is associated with the a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit. J. Biol. Chem. 273, 19518-19524

57. Mehta, S., Wu, H., Garner, C. C., and Marshall, J. (2001) Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97. J. Biol. Chem. 276, 16092-16099

58. Cai, C., Coleman, S. K., Niemi, K., and Keinanen, K. (2002) Selective binding of synapse-associated protein 97 to GluR-A a-amino-5-hydroxy-3-methyl-4-isoxazole-4-propionate receptor subunit is determined by a novel sequence motif. J. Biol. Chem. 277, 31484-31490

59. Hall, D. D., Davare, M. A., Shi, M., Allen, M. L., Weisenhaus, M., McKnight, G. S., and Hell, J. W. (2007) Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons. Biochemistry. 46, 1635-1646

60. Leonard, A. S., and Hell, J. W. (1997) Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites. J. Biol. Chem. 272, 12107-12115

61. Leonard, A. S., Lim, I. A., Hemsworth, D. E., Horne, M. C., and Hell, J. W. (1999) Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. U. S. A. 96, 3239-3244

62. Valtschanoff, J. G., Burette, A., Davare, M. A., Leonard, A. S., Hell, J. W., and Weinberg, R. J. (2000) SAP97 concentrates at the postsynaptic density in cerebral cortex. Eur. J. Neurosci. 12, 3605-3614

63. Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) 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. 274, 30280-30287

64. Davare, M. A., and Hell, J. W. (2003) Increased phosphorylation of the neuronal L-type Ca(2+) channel Ca(v)1.2 during aging. Proc. Natl. Acad. Sci. U. S. A. 100, 16018-16023

65. Hall, D. D., Feekes, J. A., Arachchige Don, A. S., Shi, M., Hamid, J., Chen, L., Strack, S., Zamponi, G. W., Horne, M. C., and Hell, J. W. (2006) Binding of protein phosphatase 2A to the L-type calcium channel Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928 dephosphorylation. Biochemistry. 45, 3448-3459

66. Boehm, J., Kang, M. G., Johnson, R. C., Esteban, J., Huganir, R. L., and Malinow, R. (2006) Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1. Neuron. 51, 213-225

67. Vanhoose, A. M., and Winder, D. G. (2003) NMDA and beta1-adrenergic receptors differentially signal phosphorylation of glutamate receptor type 1 in area CA1 of hippocampus. J. Neurosci. 23, 5827-5834

68. Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T., and Huganir, R. L. (1997) Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J. Biol. Chem. 272, 5157-5166

at University of C

alifornia, Davis, on M

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.jbc.orgD

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69. Westphal, R. S., Tavalin, S. J., Lin, J. W., Alto, N. M., Fraser, I. D. C., Langeberg, L. K., Sheng, M., and Scott, J. D. (1999) Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science. 285, 93-96

70. Fraser, I. D., Cong, M., Kim, J., Rollins, E. N., Daaka, Y., Lefkowitz, R. J., and Scott, J. D. (2000) Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr. Biol. 10, 409-412

71. Dai, S., Hall, D. D., and Hell, J. W. (2009) Supramolecular Assemblies and Localized Regulation of Voltage-gated Ion Channels. Physiol. Rev. 89, 411-452

72. Buzsaki, G. (2002) Theta oscillations in the hippocampus. Neuron. 33, 325-340 73. Mizuseki, K., Sirota, A., Pastalkova, E., and Buzsaki, G. (2009) Theta oscillations provide temporal

windows for local circuit computation in the entorhinal-hippocampal loop. Neuron. 64, 267-280 74. Thomas, M. J., Moody, T. D., Makhinson, M., and O'Dell, T. J. (1996) Activity-dependent beta-

adrenergic modulation of low frequency stimulation induced LTP in the hippocampal CA1 region. Neuron. 17, 475-482

75. Katsuki, H., Izumi, Y., and Zorumski, C. F. (1997) Noradrenergic regulation of synaptic plasticity in the hippocampal CA1 region. J. Neurophysiol. 77, 3013-3020

76. Gelinas, J. N., and Nguyen, P. V. (2005) Beta-adrenergic receptor activation facilitates induction of a protein synthesis-dependent late phase of long-term potentiation. J. Neurosci. 25, 3294-3303

77. Makino, Y., Johnson, R. C., Yu, Y., Takamiya, K., and Huganir, R. L. (2011) Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor. Proc. Natl. Acad. Sci. U. S. A. 108, 8450-8455

78. Adesnik, H., and Nicoll, R. A. (2007) Conservation of glutamate receptor 2-containing AMPA receptors during long-term potentiation. J. Neurosci. 27, 4598-4602

79. Plant, K., Pelkey, K. A., Bortolotto, Z. A., Morita, D., Terashima, A., McBain, C. J., Collingridge, G. L., and Isaac, J. T. (2006) Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat. Neurosci. 9, 602-604

80. Bellone, C., and Luscher, C. (2006) Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression. Nat. Neurosci. 9, 636-641

81. Clem, R. L., Celikel, T., and Barth, A. L. (2008) Ongoing in vivo experience triggers synaptic metaplasticity in the neocortex. Science. 319, 101-104

82. Clem, R. L., and Huganir, R. L. (2010) Calcium-permeable AMPA receptor dynamics mediate fear memory erasure. Science. 330, 1108-1112

83. Thiagarajan, T. C., Lindskog, M., and Tsien, R. W. (2005) Adaptation to synaptic inactivity in hippocampal neurons. Neuron. 47, 725-737

84. Isaac, J. T., Ashby, M. C., and McBain, C. J. (2007) The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron. 54, 859-871

85. Magazanik, L. G., Buldakova, S. L., Samoilova, M. V., Gmiro, V. E., Mellor, I. R., and Usherwood, P. N. (1997) Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by adamantane derivatives. J. Physiol. 505 ( Pt 3), 655-663

86. Gray, E. E., Fink, A. E., Sarinana, J., Vissel, B., and O'Dell, T. J. (2007) Long-term potentiation in the hippocampal CA1 region does not require insertion and activation of GluR2-lacking AMPA receptors. J. Neurophysiol. 98, 2488-2492

87. Guire, E. S., Oh, M. C., Soderling, T. R., and Derkach, V. A. (2008) Recruitment of calcium-permeable AMPA receptors during synaptic potentiation is regulated by CaM-kinase I. J. Neurosci. 28, 6000-6009

88. Havekes, R., Canton, D. A., Park, A. J., Huang, T., Nie, T., Day, J. P., Guercio, L. A., Grimes, Q., Luczak, V., Gelman, I. H., Baillie, G. S., Scott, J. D., and Abel, T. (2012) Gravin Orchestrates Protein Kinase A and beta2-Adrenergic Receptor Signaling Critical for Synaptic Plasticity and Memory. J. Neurosci. 32, 18137-18149

at University of C

alifornia, Davis, on M

ay 8, 2013w

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89. Shih, M., Lin, F., Scott, J. D., Wang, H. Y., and Malbon, C. C. (1999) Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin. J. Biol. Chem. 274, 1588-1595

90. Lin, F., Wang, H., and Malbon, C. C. (2000) Gravin-mediated formation of signaling complexes in beta 2-adrenergic receptor desensitization and resensitization. J. Biol. Chem. 275, 19025-19034

91. Tao, J., Wang, H. Y., and Malbon, C. C. (2003) Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor. EMBO J. 22, 6419-6429

92. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature. 390, 88-91

93. Baillie, G. S., Sood, A., McPhee, I., Gall, I., Perry, S. J., Lefkowitz, R. J., and Houslay, M. D. (2003) beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi. Proc. Natl. Acad. Sci. U. S. A. 100, 940-945

94. Hell, J. W., Westenbroek, R. E., Breeze, L. J., Wang, K. K. W., Chavkin, C., and Catterall, W. A. (1996) N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 93, 3362-3367

95. Hoogland, T. M., and Saggau, P. (2004) Facilitation of L-type Ca2+ channels in dendritic spines by activation of beta2 adrenergic receptors. J. Neurosci. 24, 8416-8427

96. Lin, L., Sun, W., Kung, F., Dell'Acqua, M. L., and Hoffman, D. A. (2011) AKAP79/150 impacts intrinsic excitability of hippocampal neurons through phospho-regulation of A-type K+ channel trafficking. J. Neurosci. 31, 1323-1332

97. Hammond, R. S., Lin, L., Sidorov, M. S., Wikenheiser, A. M., and Hoffman, D. A. (2008) Protein kinase a mediates activity-dependent Kv4.2 channel trafficking. J. Neurosci. 28, 7513-7519

98. Grover, L. M., and Teyler, T. J. (1990) Two components of long-term potentiation induced by different patterns of afferent activation. Nature. 347, 477-479

99. Shankar, S., Teyler, T. J., and Robbins, N. (1998) Aging differentially alters forms of long-term potentiation in rat hippocampal area CA1. J. Neurophysiol. 79, 334-341

100. Cahill, L., Prins, B., Weber, M., and McGaugh, J. L. (1994) Beta-adrenergic activation and memory for emotional events. Nature. 371, 702-704

101. Nielson, K. A., and Jensen, R. A. (1994) Beta-adrenergic receptor antagonist antihypertensive medications impair arousal-induced modulation of working memory in elderly humans. Behav. Neural. Biol. 62, 190-200

102. Berman, D. E., and Dudai, Y. (2001) Memory extinction, learning anew, and learning the new: dissociations in the molecular machinery of learning in cortex. Science. 291, 2417-2419

103. Strange, B. A., Hurlemann, R., and Dolan, R. J. (2003) An emotion-induced retrograde amnesia in humans is amygdala- and beta-adrenergic-dependent. Proc. Natl. Acad. Sci. U. S. A. 100, 13626-13631

104. Strange, B. A., and Dolan, R. J. (2004) Beta-adrenergic modulation of emotional memory-evoked human amygdala and hippocampal responses. Proc. Natl. Acad. Sci. U. S. A. 101, 11454-11458

105. Minzenberg, M. J., Watrous, A. J., Yoon, J. H., Ursu, S., and Carter, C. S. (2008) Modafinil shifts human locus coeruleus to low-tonic, high-phasic activity during functional MRI. Science. 322, 1700-1702

106. Carter, M. E., Yizhar, O., Chikahisa, S., Nguyen, H., Adamantidis, A., Nishino, S., Deisseroth, K., and de Lecea, L. (2010) Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat. Neurosci. 13, 1526-1533

107. Lin, Y. W., Min, M. Y., Chiu, T. H., and Yang, H. W. (2003) Enhancement of associative long-term potentiation by activation of beta-adrenergic receptors at CA1 synapses in rat hippocampal slices. J. Neurosci. 23, 4173-4181

108. Walling, S. G., and Harley, C. W. (2004) Locus ceruleus activation initiates delayed synaptic potentiation of perforant path input to the dentate gyrus in awake rats: a novel beta-adrenergic- and protein synthesis-dependent mammalian plasticity mechanism. J. Neurosci. 24, 598-604

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FOOTNOTES *This work was supported by the NIH grants NS078792 (JWH) and AG017502 (JWH) and a presidential predoctoral fellowship from the University of Iowa (MZ). 12-471 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242 2To whom correspondence should be addressed: 451 E Health Sciences Drive, Davis, CA 95616-8636. Phone: (530) 752 6540; FAX: (530) 752 7710; E-mail: [email protected]. FIGURE LEGENDS FIGURE 1. Overview of the relevant interactions within the GluA1/A2/γ - PSD-95 - β2 AR - SAP97 - AKAP5/AC/PP2B/PKA complex. AKAP5 (green) recruits PKA and the antagonistic phosphatase PP2B to GluA1 for dynamic phosphorylation and dephosphorylation of S845 (17,37,42,48-50). In detail, a central not completely defined region of AKAP5 interacts with the SH3 and GK domains of PSD-95 and SAP97 (blue) (48,51). SAP97 binds with its first and second PDZ domains to the C-terminus of GluA1 (56-58) and PSD-95 binds with its first and second PDZ domains to stargazin (γ2) and its homologues γ3, γ4, and γ8 (collectively depicted as γ), which in turn associate with AMPARs (52,54,55). The β2 AR binds with its very C-terminus to the third PDZ domain of PSD-95(5,6). Various ACs can directly bind with their divergent N-termini to AKAP5; AC5 and AC6 but likely not AC2 and AC9 bind to the second of the three poly-basic regions in the N-terminus of AKAP5 (10-12). FIGURE 2. AKAP5 links ACs to GluA1. Forebrains from WT, AKAP5 D36, and AKAP5 KO mice were extracted with Triton X-100 and cleared of non-solubilized material by ultracentrifugation. A, C, E. Lysate samples underwent IP with 1 μg of antibody against GluA1 or GluN1 or 1 μg control rabbit IgG (A, E) or were directly applied (C) to immunoblotting with a pan-specific antibody against all ACs and with antibodies against GluA1, RIIα, or AC5/6 as indicated. B, D, F. Immunosignals were quantified by densitometry. Depicted are film optical density (OD) ratios for panAC vs. GluA1 signals (B), OD values for panAC (D), and OD ratios for AC5/6 signals in GluN1 vs. in GluA1 IPs (F). Co-IP of ACs with GluA1 is nearly completely absent in GluA1 KO mice but not affected in D36 mice (** p<0.01, one way ANOVA). Co-IP of AC5/6 with GluN1 is nearly undetectable compared to GluA1 IPs (** p<0.01, t-test). FIGURE 3. AKAP5-anchored AC is required for phosphorylation of GluA1 on S845 upon β2 AR stimulation. A, B. Acute forebrain slices from 8-12 week old WT C57BL/6, AKAP5 D36, and AKAP5 KO mice were incubated with vehicle, ISO (10 μM), or ISO plus propranolol (1 μM; Prop) for 5 min before extraction, IP of GluA1 and immunoblotting with antibodies against phospho-S845 (pS845), phospho-S831 (pS831) and total GluA1 (GluA1; A) or IP of the NMDA receptor with an antibody against the GluN2B subunit and immunoblotting with antibodies against phospho-S897 (pS897), and total GluN1 (GluN1; B). IgG refers to control IPs with non-specific rabbit IgG to ensure specificity of GluA1 and GluN2B IPs. C, D. Immunosignals were quantified for phospho-S845, phospho-S831, and phospho-S897 and corrected for variations in total GluA1 and GluN1 loading. Graphed are averages ± SEM of relative phosphorylation levels under basal conditions (C) and averages ± SEM of fold increases by ISO and ISO+Propranolol (Prop) vs. basal phosphorylation levels (D) for each genotype (bottom bar diagrams). The number n in inserts on upper right part of C indicates number of independent experiments (for each experiment one mouse per genotype was used; * p<0.05, ** p<0.01, *** p<0.001 for KO or D36 vs. WT). ISO significantly increased GluA1 S845 and GluN1 S897 phosphorylation and propranolol antagonized these increases in all 3 genotypes (the statistically significant treatment effects are not depicted in the graphs for simplicity). Neither treatment affected S831 phosphorylation in any genotype. Statstical analysis: Basal GluA1 S845 phosphorylation was decreased for KO but not D36 vs. WT (one way ANOVA: p=0.0375; Tukey’s multiple comparison test for WT vs KO is p<0.05 and for WT vs D36 is p>0.05). ISO-induced fold increase of S845 phosphorylation was decreased for KO and for D36 vs.

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WT (two-way ANOVA p=0.0243 for genotype and p=0.0012 for treatments with ISO and ISO+Prop; Bonferroni post test for ISO treatment for WT vs. KO p<0.01 and for WT vs. D36, p<0.001; p>0.05 for both comparisons for ISO+Prop treatment). Basal GluA1 S831 phosphorylation was unaltered in KO and D36 vs. WT mice (one way ANOVA p=0.2639). ISO did not increase S831 phosphorylation in any of the three genotypes (two way ANOVA p=0.8826 for genotypes and p=0.2658 for treatments). The slight decrease in basal GluN1 S897 phosphorylation in KO and D36 vs. WT was statistically not significant (one way ANOVA: p=0.2242). ISO-induced increases in S897 phosphorylation were comparable for all three genotypes (two way ANOVA p=0.7098 for genotypes and p=0.0001 for treatments). FIGURE 4. AKAP5 is dispensable for the complex formation between β2 AR and GluA1. Forebrain slices from WT, AKAP5 D36, AKAP5, and β2 AR KO mice were extracted with Triton X-100 before IP with 8 μg of the H-20 antibody against β2 AR, the V-19 antibody against β1 AR, or non-immune rabbit IgG. A. Forebrain slices from WT, AKAP5 D36, and AKAP5 KO mice were extracted with Triton X-100 before IP with 8 μg of the H-20 antibody against β2 AR and immunoblotting for GluA1, GluA2, AKAP5, PSD-95, and the β2 AR itself. A mix of 1/3 WT, 1/3 D36 and 1/3 KO extract was used for control IP with 8 μg of non-immune rabbit IgG (left lane). B. Immunosignals from A were quantified by densitometry. The ratio of GluA1 to β2 AR from 4 independent experiments was quantified and normalized to the WT signal from the same experiment. There is no statistically significant difference between genotypes for any of these parameters except for lack of AKAP5 in AKAP5 KO mice. C,D. Forebrains from WT and β2 AR KO mice were extracted with Triton X-100 before IP of β2 AR, β1 AR (V-19 antibody), or non-immune rabbit IgG. C. Immunoblotting for β1 AR and, after stripping, β2 AR shows respective isoform specificity of each antibody. Similar results were obtained in 2 other experiments. D. Immunoblotting after IP with the β2 AR antibody shows presence of AC5/6, AKAP5, and GluA1 only in IPs from WT but not β2 AR KO mice. Similar results were obtained in 2 other experiments. FIGURE 5. AKAP5 is not required for association of the β2 AR with PSD. A, C. 10 μg of crude lysate (Lys), P2, synaptosomal-enriched (Syn), and PSD fractions from WT (W) and either AKAP5 KO mice (K; A) or D36 mice (D; C) were separated by SDS-PAGE before immunoblotting for the indicated proteins. B, D. PSD signals for each protein from D36 (B) or KO mice (D) were quantified and normalized to the respective PSD-95 signal for each sample. C, E. AC5/6 signals in Syn and PSD fractions from WT and AKAP5 KO mice were quantified and compared to each other. The data represent the average ± SEM for n independent experiments (** p<0.01, *** p<0.001, t test). FIGURE 6. AKAP5 is necessary for GluA1 S845 phosphorylation upon β2 AR stimulation in the hippocampus. A. Acute hippocampal slices from 8-12 week old WT C57BL/6 and AKAP5 KO mice were incubated with vehicle or ISO (10 μM) for 5 min before solubilization, IP of GluA1, immunoblotting with antibodies against phospho-S845 (pS845), stripping and re-probing for phospho-S831 (pS831) and ultimately total GluA1 (GluA1). B, C. Immunosignals were quantified for phospho-S845 and phospho-S831 and corrected for variations in total GluA1 content. Graphed are averages ± SEM of relative phosphorylation levels (* p<0.05, two way ANOVA).

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FIGURE 7. ISO-induced increases in basal synaptic transmission and PTT-LTP by a 5 Hz / 3 min tetanus in the presence of ISO are impaired in AKAP5 D36 and KO mice. A-D. Time courses of fEPSPs before and after perfusion with ISO (1 µM; upper grey bar) and delivery of the tetanus (lower black bar) from recordings without (A) and with (B) an ISO baseline response in WT slice and from D36 (C) and KO (D) slices. Shown are averages of initial slopes of fEPSP starting after baseline had stabilized. Inserts on top: examples of fEPSPs before ISO application (dashed lines), after the start of ISO application (grey lines) and after PTT-LTP induction (solid lines). Graphed are averages of 10 consecutive fEPSPs recorded at the indicated times (arrows). E. Summary data of PTT-LTP. The baseline (Bsl) is the average of the fEPSP initial slopes from each individual experiment during the 5 min immediately preceding start of the ISO application and equaled 100% for each experiment. The 5Hz/3min tetanus induced PTT-LTP in WT Group 1 (p=0.0002; t-test), WT Group 2 (p=0.0464), and D36 (p<0.0001) but not AKAP5 KO (p=0.943; not depicted in diagram for simplicity). Compared to the interleaved WT recordings, the direct ISO effects as well as PTT-LTP levels were significantly lower for D36 and KO mice. Two way ANOVA showed a genotype effect: p<0.0001; treatment effect (PTT-LTP induction): p<0.0001; ISO and PTT-LTP effects between genotypes are indicated in bar graphs (*p <0.05; **p <0.01; ***p <0.001). Accordingly, PTT-LTP is significantly reduced in D36 vs. WT and significantly more reduced (in fact abolished) in KO. FIGURE 8. ISO-induced increases in basal synaptic transmission depend on β2 AR but not β1 AR. A,B. Time courses of fEPSPs before and after perfusion with ISO (1 µM; lower grey bar) in the presence of 40 nM ICI118551 (A) or 1 μM CGP20712 (B). Shown are averages of initial slopes of fEPSP starting after baseline had stabilized. Inserts on bottom: examples of fEPSPs before (black lines) and after the start of ISO application (grey lines). Graphed are averages of 10 consecutive fEPSPs recorded at the indicated times (arrows). C. Summary data. The baseline is the average of the fEPSP initial slopes from each individual experiment during the 5 min immediately preceding start of the ISO application and equaled 100% for each experiment. The ISO bars show the increase in fEPSP responses, which were obtained by averaging the initial slope values measured 10-15 min after the onset of ISO perfusion. ISO did not induce any increase in fEPSPs in any of the 6 slices test in the presence of ICI118551 (p=0.3179; t-test) but increased fEPSPs in the 3 slices tested in the presence of CGP20712 (p=0.0297 depicted by *; t-test). FIGURE 9. Induction of PTT-LTP requires GluA2-lacking AMPA receptors. A. Time course of fEPSPs before and after perfusion with ISO (1 µM; grey bar) in the presence of IEM1460 (30 µM; upper black bar) and delivery of the tetanus (lower black bar). Shown are averages of initial slopes of fEPSP starting after baseline had stabilized. Inserts on top: examples of fEPSPs immediately before (left) and 30 min after delivery of tetani (right). B, C. Time course of fEPSPs in slices perfused with IEM1460 (30 µM; black bar in B) or without this drug (C) before and after delivery of the two 100 Hz/1 sec tetani, which were 10 sec apart (arrowheads indicate start of first tetani). Inserts on top: examples of fEPSPs immediately before (left) and 45 min after LTP induction (right). D. Summary data of IEM1460 vs interleaved control experiments. For each experiment the averages of the fEPSP initial slope values over the 5 min immediately preceding the tetani constitute the baseline corresponding to 100 %. The bars show the increase in fEPSP responses, which were obtained by averaging the initial slope values measured 40-45 min after the tetani. Illustrated on the left are the averages for PTT-LTP in the presence of ISO only and ISO + IEM1460 (p<0.01 for PTT-LTP without vs. with IEM1460; depicted by **; two way ANOVA) and on the right for 2x100 Hz LTP without and with IEM1460 present (p=0.7745; two way ANOVA). IEM1460 clearly blocked PTT-LTP but had no effect on 2x100 Hz LTP.

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PropIsoProp porPosI Iso

pS83

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crea

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pS89

7(fo

ld in

crea

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0

2

4

6

8

0

2

4

6

0

1

2

3

**

3

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WT D36 KO W

T D36 KOIgG

β2ARIP

AKAP5

GluA1

GluA2

PSD-95

β2AR

A

BAKAP5

0

50

100

150

/ P

5A

KA

βR

A2

WT D36 KO

n 44 4

GluA2

0

50

100

/ 2Aul

Gβ

RA2

WT D36 KO

n 4 4 4

/ 1Aul

Gβ

RA2

GluA1

0

50

100

WT D36 KO

4 4 4n

PSD-95

0

50

100

/ 59-D

SP

βR

A2

WT D36 KO

4 4 4n

4

AC 5/6

GluA1

β2AR

β2AR

IgG IgG

WT β2

AR KO

IP

AKAP5

β1 AR

β2 AR

β1 AR

β2 AR

IgGIP :C

D

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alifornia, Davis, on M

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A C

B D

W KW KW KW K

GluA1

β2AR

PSD-95

Synaptophysin

Lys P2 Syn PSD

AKAP5W D W DW D W D

GluA1

β2AR

PSD-95

Synaptophysin

Lys P2 Syn PSD

AKAP5

β1AR

5

ACAC

β1AR

PKA RIIα PKA RIIα

Pro

tein

/ P

SD

95

0

50

100

150

n= 5 3 5 5

AKAP5 KO

*****

GluA1 β2ARAC RIIα

/ nietorP

59D

SP

GluA1 β2ARAC0

50

100

150

n= 4 4 6 5

AKAP5 D36

**

RIIα

KO WT KO WTSYN PSD

AC 5/6

AC 5/6

WT KO

WT KO

0

50

100

150

**

Sig

nal I

nten

sity

%

n 3 333

SYNPSD

E F

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WT KO

pS845

pS831

IP: GluA1

- + - +

GluA1

ISO

CtrIS

O CtrIS

O0

50

100

150

200 WTAKAP5 KO

*

pS84

5 / G

luA1

Sig

nal %

CtrIS

O CtrIS

O0

50

100

150WTAKAP5 KO

pS83

1 / G

luA1

Sig

nal %

n 5 5 5 5

5 5 5 5n

C

6

A

B

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alifornia, Davis, on M

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Time (min)

fEPS

P slo

pe(%

bas

elin

e)

-20 -10 0 10 20 30 40 50

60

80

100

120

140

160

180

Time (min)

fEPS

P slo

pe(%

bas

elin

e)

-20 -10 0 10 20 30 40 50

60

80

100

120

140

160

180

Time (min)

fEPS

P slo

pe(%

bas

elin

e)

-20 -10 0 10 20 30 40 50

60

80

100

120

140

160

180

Time (min)

fEPS

P slo

pe(%

bas

elin

e)

-20 -10 0 10 20 30 40 50

60

80

100

120

140

160

180

50

100

150

200

250 ISOISO+5Hz

Bsl WTGroup 13 mice5 slices

WTGroup 25 mice7 slices

AKAPD36

7 mice9 slices

AKAPKO

3 mice6 slices

fEPS

P slo

pe(%

bas

elin

e)

Bsl,

10 ms

0.5

mV ISO,

ISO + 5Hz,

A WT group 1 B WT group 2

C AKAP D36 D AKAP KO

E

Bsl,

10 ms

0.5

mV ISO,

ISO + 5Hz,

****

****

ISO 1μM 5Hz

ISO 1μM 5Hz

ISO 1μM 5Hz

ISO 1μM 5Hz

Bsl,

ISO + 5Hz, ISO,

10 ms

0.5

mV

Bsl,

ISO,

10 ms

0.5

mV

ISO + 5Hz,

7

**

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A

B

C

-5 0 5 10 15

60

80

100

120

140

Time (min)

fEPS

P sl

ope

(% b

asel

ine)

ISO 1 μM

ICI 40 nM

-5 0 5 10 15

60

80

100

120

140

Time (min)

fEPS

P sl

ope

(% b

asel

ine)

ISO 1 μM

CGP 1 μM

0

50

100

150

fEPS

P sl

ope

(% b

asel

ine)

ICI,ICI+ISO,

10 ms

0.5

mV

10 ms0.5

mV

CGP,CGP+ISO,

CGPICI

*ISObaseline

n 36 6 3

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A B

D

PTT-LTP 100 Hz

10 ms0.5

mV

-15 0 15 30 450

50

100

150

200

Time (min)

fEPS

P (%

of b

asel

ine)

Vehicle

IEM14

60

-15 0 15 30 450

50

100

150

200

Time (min)fE

PSP

(% b

asel

ine)

fEPS

P (%

Base

line)

ISO IS

O

+ IEM14

6050

100

150

200 **

IEM1460

ISO

IEM1460

5 Hz / 180 s 100 Hz / 1 s

ISO: 7 mice, 12 slicesISO + IEM1460: 5 mice, 8 slicesVehicle: 6 mice, 11 slicesIEM1460: 5 mice, 7 slices

5 Hz, 3 min

100 Hz, 1 sec, 2x

-15 0 15 30 450

50

100

150

200

Time (min)

fEPS

P (%

Base

line)

C

100 Hz, 1 sec, 2x

9 at U

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