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NMDA Receptor-Dependent GABAB Receptor Internalization Via Camkii Phosphorylation of Serine 867 in GABAB1

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NMDA Receptor-Dependent GABAB Receptor Internalization Via Camkii Phosphorylation of Serine 867 in GABAB1 NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1 Nicole Guetga,1, Said Abdel Aziza,1, Niklaus Holbrob, Rostislav Tureceka,c, Tobias Roseb, Riad Seddika, Martin Gassmanna, Suzette Moesd, Paul Jenoed, Thomas G. Oertnerb, Emilio Casanovaa,2, and Bernhard Bettlera,3 aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland; bFriedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; cInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic; and dBiozentrum, University of Basel, 4056 Basel, Switzerland Edited* by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 24, 2010 (received for review January 22, 2010) 2+ GABAB receptors are the G-protein–coupled receptors for GABA, the (13, 14) and reduces Ca permeability (15) of NMDA receptors. main inhibitory neurotransmitter in the brain. GABAB receptors are Reciprocally, there is evidence that glutamate receptors decrease abundant on dendritic spines, where they dampen postsynaptic ex- surface expression of GABAB receptors (16–18). This supports 2+ fl citability and inhibit Ca in ux through NMDA receptors when acti- that glutamate receptors and GABAB receptors cross-talk in vated by spillover of GABA from neighboring GABAergic terminals. dendrites and spines. Here, we show that an excitatory signaling cascade enables spines Neither the glutamate receptors nor the signaling pathways to counteract this GABAB-mediated inhibition. We found that NMDA controlling surface availability of GABAB receptors have yet been application to cultured hippocampal neurons promotes dynamin- identified. Here we show that NMDA receptor-dependent phos- dependent endocytosis of GABAB receptors. NMDA-dependent inter- phorylation via CaMKII targets GABAB receptors for in- 2+ nalization of GABAB receptors requires activation of Ca /Calmodulin- ternalization. This postsynaptic regulation of GABAB receptors 2+ dependent protein kinase II (CaMKII), which associates with GABAB has implications for the control of local excitability and Ca - receptors in vivo and phosphorylates serine 867 (S867) in the intracel- dependent neuronal functions. NEUROSCIENCE lular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to Results NMDA-mediated internalization. Time-lapse two-photon imaging of NMDA Receptors Mediate GABAB Receptor Internalization. We used organotypic hippocampal slices reveals that activation of NMDA transfected cultured hippocampal neurons to identify the gluta- receptors removes GABAB receptors within minutes from the surface mate receptors regulating cell surface expression of GABAB of dendritic spines and shafts. NMDA-dependent S867 phosphoryla- receptors. Robust cell surface expression of tagged GABAB1b tion and internalization is predominantly detectable with the subunits (HA-GB1b-eGFP) was observed upon cotransfection GABAB1b subunit isoform, which is the isoform that clusters with in- with GABAB2 subunits (Fig. 1A), which are mandatory for + hibitory effector K channels in thespines. Consistentwith this, NMDA GABAB1 surface expression (8, 19, 20). GABAB1b surface ex- receptor activation in neurons impairs the ability of GABAB receptors pression was monitored by immunolabeling of the extracellular to activate K+ channels. Thus, our data support that NMDA receptor HA-tag before permeabilization of cells (red fluorescence). Total activity endocytoses postsynaptic GABAB receptors through CaMKII- GABAB1b expression was monitored by immunolabeling of the mediated phosphorylation of S867. This provides a means to spare intracellular eGFP-tag after permeabilization of cells (green NMDA receptors at individual glutamatergic synapses from re- fluorescence). To quantify the level of surface GABAB1b protein, ciprocal inhibition through GABAB receptors. we calculated the ratio of red to green fluorescence intensity (Fig. 1 B and C). Upon glutamate treatment (50 μM glutamate/5 μM γ fi -aminobutyric acid | spines | traf cking | synaptic plasticity | GABAB glycine for 30 min), surface GABAB1b protein was significantly reduced (41.4 ± 5.3% of control, n = 10, P < 0.001), consistent ABAB receptors modulate the excitability of neurons with published data (18). The NMDA receptor antagonist APV throughout the brain. They are therapeutic targets for a vari- (100 μM for 2 h) prevented the glutamate-induced decrease in G ± n P > ety of disorders, including cognitive impairments, addiction, anx- surface GABAB1b protein (98.4 12.6% of control, =9, iety, depression, and epilepsy (1). Depending on their subcellular 0.05). We tested whether a selective activation of NMDA recep- tors is sufficient to decrease surface GABA protein. Following localization GABAB receptors exert distinct regulatory effects on B1b NMDA treatment (75 μM NMDA/5 μM glycine for 3 min) and synaptic transmission (2–4). Presynaptic GABAB receptors inhibit neurotransmitter release (5, 6). Postsynaptic GABA receptors recovery in conditioned medium for 27 min, surface GABAB1b B fi ± n dampen neuronal excitability by gating Kir3-type K+ channels, protein was signi cantly reduced (54.8 3.2% of control, =10, which generates slow inhibitory postsynaptic potentials and local shunting (7). Molecular diversity in the GABAB system arises from the GABA and GABA subunit isoforms, both of which Author contributions: N.G., S.A.A., N.H., R.T., T.R., R.S., M.G., P.J., E.C., and B.B. designed B1a B1b research; N.G., S.A.A., N.H., R.T., T.R., R.S., S.M., and E.C. performed research; T.G.O. combine with GABAB2 subunits to form heteromeric GABAB(1a,2) contributed new reagents/analytic tools; N.G., S.A.A., N.H., R.T., T.R., R.S., M.G., S.M., and GABAB(1b,2) receptors (8). Genetically modified mice re- P.J., and E.C. analyzed data; and N.G., M.G., and B.B. wrote the paper. vealed that the two receptors convey nonredundant synaptic The authors declare no conflict of interest. functions at glutamatergic synapses, owing to their differing dis- *This Direct Submission article had a prearranged editor. tribution to axonal and dendritic compartments (3, 9). Selectively 1N.G. and S.A.A. contributed equally to this work. GABAB(1a,2) receptors control the release of glutamate, whereas 2Present address: Ludwig Boltzmann Institute for Cancer Research, A-1090 Vienna, predominantly GABAB(1b,2) receptors activate postsynaptic Austria. Kir3 channels in dendritic spines (10–12). Activation of GABAB 3To whom correspondence should be addressed. E-mail: [email protected]. receptors on spines inhibits NMDA receptors through hyperpo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ larization and the PKA pathway, which enhances Mg block 1073/pnas.1000909107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000909107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A B A ** C ) ** ** input Co-IP surface GB1b total GB1b overlay ** * ) 100 100 serum gp anti-GB1 anti-GB2 serum rb I normalized 50 control CaMKI I surface GB1b (% 50 anti- 0 CaMKI control glutamate normalized glutamate NMDA surface GB1b (% D + APV 0 -GB1 control NMDA -GB2 glutamate * NMDA+EGTA-AM NMDA+KN93 beads beads + GST beads beads + GST C NMDA+KN92 + GST ) * I 100 CaMKI I 3 anti- B CaMKI 50 NMDA NMDA + KN9 control normalized glutamate+APV E surf surface GB1b (% 0 GB1a +CaMKII -CaMKII control NMDA 1 2 2 GB1b 1 NMDA dynasore GB + dynasore total NMDA T- GB T- GB T- GB1a T- GB NMDA anti-GB1 GS T GS GS GS GS GS + CGP54626A GB1b T GS surf GS T- GB 2 TP Fig. 1. NMDA-dependent removal of surface GABA receptors. (A) Rat hippo- GB 2 GS T- GB 1 B P]A total campal neurons coexpressing exogenous HA-GB1b-eGFP and GABAB2 were 32 γ [- GS T anti-GB2 GB 2 treated at DIV14 as indicated. Surface GABAB1b (GB1b) protein was fluorescence- e GS T- GB 2 labeled with anti-HA antibodies before permeabilization. Total GB1b protein surf GS T- GB 1 was fluorescence-labeled with anti-eGFP antibodies after permeabilization. tub total Single optical planes captured with a confocal microscope are shown. (Scale bar, coomassi GS T tub 15 μm.) (Insets) Representative spines at higher magnification. (B) Surface anti-tubulin GABAB1b protein was quantified by the ratio of surface to total fluorescence intensity. Values were normalized to control values in the absence of any phar- Fig. 2. NMDA-induced removal of surface GABAB receptors requires CaM- KII. (A) Rat hippocampal neurons coexpressing exogenous HA-GB1b-eGFP macological treatment. Surface GABAB1b protein was significantly decreased following glutamate or NMDA treatment. No significant reduction was observed and GABAB2 were analyzed at DIV14. Surface GABAB1b protein was quan- n – P < tified by the ratio of surface to total fluorescence intensity. Preincubation of with glutamate treatment after preincubation with APV. =9 10, ** 0.01, 2+ ***P < 0.001. (C) Dynasore but not CGP54626A prevented the NMDA-induced neurons with the Ca -chelator EGTA-AM or the CaMKII inhibitor KN-93 reduction of surface GABA protein. n =8–10, *P < 0.05. Quantification was prevented the NMDA-induced reduction in surface GABAB1b protein. KN-92 B1b ± n – P < B from nonsaturated images. Data are presented as mean ± SEM. was ineffective. Data are means SEM, =910, ** 0.01. ( ) NMDA- mediated removal of endogenous surface GABAB receptors. Live cortical neurons were treated at DIV14 as indicated and then biotinylated. Cell homogenates (total) and avidin-purified cell surface proteins (surf) were P < 0.01). Heteromerization with GABAB2 is mandatory for exit of probed on Western blots with anti-GABAB1 (anti-GB1) and anti-GABAB2 GABAB1 from the endoplasmic reticulum and for receptor function (anti-GB2) antibodies. While all GABAB subunits were removed from the cell (19, 20). As expected from the assembly with GABAB1, surface surface in response to NMDA, GABA was more efficiently removed than fi B1b GABAB2 protein was also signi cantly decreased following gluta- GABAB1a (P < 0.05). NMDA-mediated removal of surface protein was mate or NMDA application, and this decrease was prevented by inhibited by KN-93.
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