Receptor for activated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABA(A) receptors with the activation of G-protein-coupled receptors

Abstract

GABA(A) receptors are the principal sites of fast synaptic inhibition in the brain. These receptors are hetero-pentamers that can be assembled from a number of subunit classes: alpha(1-6), beta(1-3), gamma(1-3), delta(1), epsilon, theta;, and pi, but the majority of receptor subtypes is believed, however, to be composed of alpha, beta, and gamma2 subunits. A major mechanism for modulating GABA(A) receptor function occurs via the phosphorylation of residues within the intracellular domains of receptor subunits by a range of serine/threonine and tyrosine kinases. However, how protein kinases are targeted to these receptors to facilitate functional modulation remains unknown. Here we demonstrate that the receptor for activated C kinase (RACK-1) and protein kinase C (PKC) bind to distinct sites on GABA(A) receptor beta subunits. Although RACK-1 is not essential for PKC binding to GABA(A) receptor beta subunits, it enhances the phosphorylation of serine 409, a residue critical for the phospho-dependent modulation of GABA(A) receptor function in the beta1 subunit by anchored PKC. Furthermore, RACK-1 also enhances GABA(A) receptor functional modulation in neurons by a PKC-dependent signaling pathway with the activation of muscarinic acetylcholine receptors (mAChRs). This PKC-dependent modulation of neuronal GABA(A) receptors was mirrored by an increase in the phosphorylation of GABA(A) receptor beta subunits with the activation of mAChRs. Our results suggest a central role for RACK-1 in potentiating PKC-dependent phosphorylation and functional modulation of GABA(A) receptors. Therefore, RACK-1 will enhance functional cross talk between GABA(A) receptors and G-protein-coupled receptors and therefore may have profound effects on neuronal excitability.

Fig. 1.

RACK-1 and PKC bind to differing sites on the GABA_A receptor β1 subunit. A, The RACK-1 binding site was identified by using filter overlay assays. Shown is 2 μg of a series of GST fusion protein deletion constructs encoding the residues; residues 302–426 (GST-β1; lane 1), 302–365 (lane 2), 366–394 (lane 3), 395–426 (lane 4), 366–404 (lane 5), and 366–415 (lane 6) of the GABA_A receptor β1 subunit intracellular domain or GST alone (lane 7) were separated by SDS-PAGE. Then duplicate gels were transferred to a nitrocellulose membrane and incubated with [³²P]-labeled RACK-1 (right) or were stained with Coomassie blue to demonstrate equal loading of GST fusion proteins (left). The binding of RACK-1 was quantified with a PhosphorImager.B, The relative binding of PKC and RACK-1 to truncations of the β1 subunit intracellular domains as derived from overlay and pull down assays. +++, Binding similar (>95%) to control (GSTβ3 302–426); ++, 60% binding relative to control; —, no detectable binding. Similar results were obtained in four separate experiments.C, The PKC binding site was identified with GST pull down assays. GST fusion protein deletion constructs encoding residues 302–426 (GST-β1; lane 1), 302–365 (lane 2), 366–394 (lane 3), 395–426 (lane 4), 366–404 (lane 5), and 366–415 (lane 6) of the GABA_A β1 subunit intracellular domain or GST alone (lane 7) were incubated with adult rat brain extracts, washed, and separated by SDS-PAGE and immunoblotting with anti-PKC-βII.Lane 8 represents 10% of the total brain extract that was exposed to the fusion proteins. The data are representative of three independent experiments. D, PKC associates with the β1 subunit intracellular domain independently of S409. GST fusion proteins encoding the entire intracellular domain of the β1 subunit (lane 1) or the mutant fusion protein GST-β1^S409A (lane 2) or GST alone (lane 3) were incubated with adult rat brain extracts, washed, and separated by SDS-PAGE. Immunoblotting with anti-PKC-βII was performed to detect associating PKC. Lane 4represents 10% of the total brain extract that was exposed to the fusion proteins. The data are representative of three independent experiments. E, Schematic diagram of proposed distinct binding sites for RACK-1 (residues 395–404) and PKC (residues 405–415).

Fig. 2.

Disrupting RACK-1 binding to the GABA_A β1 subunit receptor reduces PKC phosphorylation of the receptor. A, Pep-GC prevents RACK-1 binding to the intracellular domain of the GABA_A receptor β1 subunit. GST-β1 was exposed to adult rat brain extracts and then alone (lane 1), with 50 nm Pep-SC (lane 2), or with 50 nm Pep-GC (lane 3). Lane 4 represents 10% of the input that was used. Bound material was blotted with an anti-PKC-βII antibody (top) or anti-RACK-1 (bottom). B, GST-β1 fusion protein was phosphorylated by associated PKC activity on S409. GST-β1 (lanes 1–4) or GST-β1^S409A(lane 5) was exposed to adult rat brain extracts. After extensive washing, the fusion proteins were subjected to kinase assays alone (lanes 1, 4, 5) or in the presence of either 0.1 μm (lane 2) or 0.01 μm(lane 3) PKC inhibitor peptide (PKC_19–36).C, Pep-GC reduced the phosphorylation of GST-β1. GST-β1 was exposed to brain lysate and then incubated with 50 nm Pep-GC (lane 2), 50 nm Pep-SC (lane 3), or buffer alone (lane 1). After extensive washing, the bound material was subjected to in vitro kinase assays. The reaction products were separated by SDS-PAGE and analyzed by autoradiography (top). Also shown is a Coomassie stain of the fusion proteins to demonstrate equal loading (bottom). The data are representative of four independent experiments. D, The changes in phosphorylation of the β1 subunit in B were quantitated by the use of a PhosphorImager. The effects of Pep-GC and Pep-SC were calculated as the change relative to the untreated sample. Pep-GC produced a 27 ± 2.1% decrease in phosphorylation of GST-β1 compared with control (p > 0.001; Student's t test; n = 4).E, Binding of PKC to GST-β1 was modulated via phosphorylation of S409. GST-β1 (lanes 1, 2) or GST alone (lanes 3, 4) was subjected to in vitro kinase assays with purified PKA with (+) or without (−) ATP before exposure to adult rat brain. The final stoichiometry of phosphorylation of the GST-β1 in these experiments was 0.8 ± 0.05 mol/mol. Samples were washed and separated by SDS-PAGE. Immunoblotting with an anti-PKC-βII antibody was used to detect the association of PKC. The data are representative of five independent experiments. The average binding of PKC to phosphorylated GST-β1 was decreased by 90 ± 5% of control (p > 0.001; Student's t test; n = 5).

Fig. 3.

GABA_A receptors form a complex with PKC and RACK-1 in HEK 293 cells and in the brain. A, Immunoprecipitation of GABA_A receptors from HEK 293 cells. Transfected cells expressing the receptor α1/β1 subunits (lanes 1, 2) or control mock-transfected cells (lane 3) were labeled with [³⁵S]methionine and lysed. Cell extracts then were immunoprecipitated with anti-β1/β3 antisera (lanes 1, 3) or control nonimmune IgG (lane 2). Precipitated material was separated by SDS-PAGE, and the receptors were visualized by autoradiography. B, Coimmunoprecipitation of RACK-1 and PKC with GABA_A receptors from HEK 293 cells. HEK 293 cells expressing receptor α1/β1 subunits (lanes 1, 2) or mock-transfected cells (lane 3) were immunoprecipitated with anti-β1/β3 (lanes 1, 3) or nonimmune IgG (lane 2). Cell extracts were immunoblotted with an anti-PKC-βII antibody (top) or with an antibody specific for RACK-1 (bottom). Lane 4 represents 20% of the material that was used for the immunoprecipitations. C, PKC and RACK-1 form complexes with GABA_A receptors in vivo. Adult rat brain extracts (1 mg of total protein, lanes 1, 3; 5 mg of total protein, lanes 2, 4) were subject to immunoprecipitation with either anti-GABA_A β1/β3 antisera (lanes 3, 4) or nonimmune IgG (lanes 1, 2). Precipitated material was separated by SDS-PAGE; then immunoblotting was performed with an antibody against PKC-βII (top) or with anti-RACK-1 antibody (bottom).

Fig. 4.

PKC modulation of GABA_A receptor activation is dependent on RACK-1. GABA-activated currents were recorded under whole-cell voltage clamp at 25°C from single HEK 293 cells expressing α1β1 GABA_A receptors at a holding potential of −50 mV. GABA (10 μm) was used to activate a near-maximal response and was applied for the durations indicated at 5, 10, 15, and 25 min after formation of the whole-cell recording mode (P + t min) either in the absence (solid lines) or presence (broken lines) of 100 nm PMA. The patch pipette solution contained the control or unsupplemented solution (A; also see Materials and Methods) or incorporated a GST fusion protein of the intracellular domain of the β1 subunit, including the RACK-1 binding site (120 μg/ml; B) or the GST fusion proteins, but not including the RACK-1 binding site, sequence 302–365 (C) and sequence 366–394 (D) at 200 μg/ml. The inhibition of peak GABA-activated currents caused by PKC activation for each protocol is indicated in E. The filled bar (mean ± SEM) represents the control 10 μm GABA-activated current at P + 1 min to which all subsequent responses to GABA in each cell were normalized. The open bar (+PMA) andshaded bars (patch pipette solution supplementation with Pep-GC, GST-320–365, or GST-366–394) represent current amplitudes measured at P + 30 min in n = 4–5 cells. Thehorizontal broken lines indicate, for reference, the mean inhibition induced by 100 nm PMA in control HEK 293 cells recorded with unsupplemented patch pipette solution (E, top). The time calibration is 5 sec, and the membrane current calibrations are 100 pA (A) and 300 pA (B–D). *Significantly different from control (p > 0.01; Student's ttest; n = 4).

Fig. 5.

Time dependence of RACK-1 peptides in modulating GABA-activated currents. HEK 293 cells expressing α1β1 GABA_A receptor subunits were exposed to 0.5 μm PMA at 25°C after the formation of the whole-cell recording mode at −50 mV holding potential. The patch pipette solution was the control solution (open circles), or the solution was supplemented with 120 μg/ml RACK-1 binding site peptide (Pep-GC;filled circles) or 120 μg/ml of a scrambled version of the same peptide (Pep-SC; open squares). The GABA-activated currents were normalized to the initial currents recorded after formation of the whole-cell recording (= 1), and PMA was applied at P + 10 min. *Significantly different from cells treated with phorbol esters alone (p > 0.05; Student'st test; n = 4).

Fig. 6.

Interaction of RACK-1 with PKC modulation of GABA-activated currents in sympathetic neurons. The bar graph represents GABA-activated currents recorded at 25°C from cultured rat sympathetic ganglionic neurons [10 d in vitro (10 DIV)] at −50 mV holding potential. Peak current amplitudes were measured to 5 μm GABA at P + 25 min after formation of the whole-cell recording mode and were normalized to the currents recorded initially on achieving the whole-cell mode. The neurons were subjected to the following conditions: GABA-activated currents were recorded with control pipette solution (filled bar) and also recorded from neurons exposed to 100 nm PMA by using control pipette solution (open bar) or by using a patch pipette solution supplemented either with 120 μg/ml RACK-1 binding site peptide (Pep-GC; shaded bar) or with 120 μg/ml of the scrambled version of the RACK-1 peptide (Pep-SC; hatched bar). *Significantly different from cells treated with phorbol esters alone (Student'st test; p > 0.05;n = 4).

Fig. 7.

mAChR activation modulates GABA-induced currents via a RACK-1-dependent mechanism. GABA-activated currents are represented as a bar graph recorded at 25°C from cultured sympathetic neurons at 6 DIV and at 30 min after the initiation of whole-cell recording at −50 mV holding potential. All currents were normalized to the response to 5 μm GABA recorded after formation of the whole-cell recording mode and represent the mean ± SEM. GABA-activated currents were recorded in control Krebs' solution with normal pipette solution (filled bar) and also after treatment with 1 μm muscarine (musc) to activate muscarinic acetylcholine receptors. The muscarine-treated cells were recorded with normal pipette solution (open bar) and after supplementation of the pipette solution with either Pep-GC (shaded bar) at 200 μg/ml or PKC inhibitor peptide (hatched bar) at 15 μg/ml. *Significantly different from cells treated with muscarine alone (Student's t test; p > 0.05;n = 4).

Fig. 8.

Muscarinic acetylcholine receptor activation facilitates PKC-dependent phosphorylation of GABA_A receptor β subunits. A, The level of phosphorylation of GABA_A receptor β3 subunit in cortical neurons (8 DIV) under basal conditions (lane 1) or treated with muscarine (1 μm) for 10 min (lane 2), 20 min (lane 3), or 30 min (lane 4) or with muscarine for 20 min in the presence of the PKC inhibitor calfostin C (500 nm; lane 5) was assessed by SDS-PAGE and immunoblotting with either P-β3 408/409 antibody (UCL39; 1:50; top blot) or β3 antibody (Tot-β3; 1:200;bottom blot), followed by incubation with¹²⁵I-anti-rabbit secondary antibody. B, The level of β3 subunit phosphorylation on S408/S409 was quantitated with a PhosphorImager. The bar graph represents the levels of β3 subunit phosphorylation on S408/S409 calculated for each treatment as a percentage of control (untreated) normalized for total β3 subunit levels under the same experimental conditions. Similar results were seen in three separate experiments.