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Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

Ivankova K, Turecek R, Fritzius T, Seddik R, Prezeau L, Comps-Agrar L, Pin JP, Fakler B, Besseyrias V, Gassmann M, Bettler B - J. Biol. Chem. (2013)

Bottom Line: Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex.Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface.We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

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Surface expression of GABAB receptors is decreased in hippocampal neurons of Kctd12−/− mice.A, cell surface biotinylation assay with DIV 13 hippocampal neurons of Kctd12−/− and control littermate mice. GABAB1 (GB1a and GB1b) and KCTD12 proteins in neuronal lysates (total) and NeutrAvidin-purified cell surface proteins (surf) were revealed on Western blots using specific antibodies. Tubulin (tub) was visualized to control for loading of the SDS-PAGE. B, graph illustrating the decrease of surface GB1a and GB1b protein in neurons of Kctd12−/− mice. Data are means ± S.E. from 6–8 experiments. *, p < 0.05. C, representative GABAB receptor-mediated K+ current responses evoked by baclofen application for 25 s to cultured hippocampal neurons of Kctd12−/− (black trace) and control littermate mice (gray trace). Holding potential −50 mV. D, bar graph shows the K+ current density at peak of the response significantly reduced in Kctd12−/− as compared with control neurons. Data are the means ± S.E., n = 14, *, p < 0.05.
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Figure 7: Surface expression of GABAB receptors is decreased in hippocampal neurons of Kctd12−/− mice.A, cell surface biotinylation assay with DIV 13 hippocampal neurons of Kctd12−/− and control littermate mice. GABAB1 (GB1a and GB1b) and KCTD12 proteins in neuronal lysates (total) and NeutrAvidin-purified cell surface proteins (surf) were revealed on Western blots using specific antibodies. Tubulin (tub) was visualized to control for loading of the SDS-PAGE. B, graph illustrating the decrease of surface GB1a and GB1b protein in neurons of Kctd12−/− mice. Data are means ± S.E. from 6–8 experiments. *, p < 0.05. C, representative GABAB receptor-mediated K+ current responses evoked by baclofen application for 25 s to cultured hippocampal neurons of Kctd12−/− (black trace) and control littermate mice (gray trace). Holding potential −50 mV. D, bar graph shows the K+ current density at peak of the response significantly reduced in Kctd12−/− as compared with control neurons. Data are the means ± S.E., n = 14, *, p < 0.05.

Mentions: Hippocampal neurons of mice express KCTD12 and KCTD16 protein (5, 6). We used a cell surface biotinylation assay to address whether down-regulation of endogenous KCTD12 protein in cultured hippocampal neurons influences GABAB receptor expression at the plasma membrane. Neurons were infected at DIV 6–7 with lentiviruses encoding either KCTD12 shRNA or control shRNA and analyzed at DIV 13. KCTD12 shRNA significantly reduced the expression of endogenous KCTD12 protein as compared with control shRNA (55.8 ± 8.1% of control, KCTD12 protein was normalized to β-tubulin protein, p < 0.05; Fig. 6A). KCTD12 knockdown with shRNA resulted in a significant decrease of surface GABAB1 protein as compared with control shRNA (GABAB1a, 58.4 ± 9.7% of control, p < 0.01; GABAB1b, 66.4 ± 10.9%, p < 0.05; Fig. 6, A and B). In agreement with the results obtained from the cell surface biotinylation experiments, we found that knockdown of KCTD12 in hippocampal neurons significantly decreases the baclofen-induced K+ current density at the peak of the response (KCTD12 shRNA, 1.32 ± 0.20 pA/pF; control shRNA, 2.27 ± 0.21 pA/pF; p < 0.01; Fig. 6, C and D). Finally, we examined cell surface expression of GABAB receptors in cultured hippocampal neurons of Kctd12−/− mice (6). The cell surface biotinylation assay demonstrated that Kctd12−/− neurons express reduced amounts of GABAB1a and GABAB1b proteins at the plasma membrane in comparison with wild-type neurons (GABAB1a, 71.5 ± 9.6% of WT; GABAB1b, 77.9 ± 9.4% of WT, p < 0.05; Fig. 7, A and B). We additionally tested GABAB responses in Kctd12−/− and wild-type neurons. The GABAB receptor-mediated K+ current density at the peak of the response was slightly but significantly decreased in Kctd12−/− as compared with wild-type neurons (Kctd12−/−, 1.70 ± 0.07 pA/pF; WT, 1.94 ± 0.07 pA/pF; p < 0.05; Fig. 7, C and D). Based on these electrophysiological results, we conclude that the presence of KCTD12 increases the maximal GABAB receptor response in cultured hippocampal neurons.


Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

Ivankova K, Turecek R, Fritzius T, Seddik R, Prezeau L, Comps-Agrar L, Pin JP, Fakler B, Besseyrias V, Gassmann M, Bettler B - J. Biol. Chem. (2013)

Surface expression of GABAB receptors is decreased in hippocampal neurons of Kctd12−/− mice.A, cell surface biotinylation assay with DIV 13 hippocampal neurons of Kctd12−/− and control littermate mice. GABAB1 (GB1a and GB1b) and KCTD12 proteins in neuronal lysates (total) and NeutrAvidin-purified cell surface proteins (surf) were revealed on Western blots using specific antibodies. Tubulin (tub) was visualized to control for loading of the SDS-PAGE. B, graph illustrating the decrease of surface GB1a and GB1b protein in neurons of Kctd12−/− mice. Data are means ± S.E. from 6–8 experiments. *, p < 0.05. C, representative GABAB receptor-mediated K+ current responses evoked by baclofen application for 25 s to cultured hippocampal neurons of Kctd12−/− (black trace) and control littermate mice (gray trace). Holding potential −50 mV. D, bar graph shows the K+ current density at peak of the response significantly reduced in Kctd12−/− as compared with control neurons. Data are the means ± S.E., n = 14, *, p < 0.05.
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Figure 7: Surface expression of GABAB receptors is decreased in hippocampal neurons of Kctd12−/− mice.A, cell surface biotinylation assay with DIV 13 hippocampal neurons of Kctd12−/− and control littermate mice. GABAB1 (GB1a and GB1b) and KCTD12 proteins in neuronal lysates (total) and NeutrAvidin-purified cell surface proteins (surf) were revealed on Western blots using specific antibodies. Tubulin (tub) was visualized to control for loading of the SDS-PAGE. B, graph illustrating the decrease of surface GB1a and GB1b protein in neurons of Kctd12−/− mice. Data are means ± S.E. from 6–8 experiments. *, p < 0.05. C, representative GABAB receptor-mediated K+ current responses evoked by baclofen application for 25 s to cultured hippocampal neurons of Kctd12−/− (black trace) and control littermate mice (gray trace). Holding potential −50 mV. D, bar graph shows the K+ current density at peak of the response significantly reduced in Kctd12−/− as compared with control neurons. Data are the means ± S.E., n = 14, *, p < 0.05.
Mentions: Hippocampal neurons of mice express KCTD12 and KCTD16 protein (5, 6). We used a cell surface biotinylation assay to address whether down-regulation of endogenous KCTD12 protein in cultured hippocampal neurons influences GABAB receptor expression at the plasma membrane. Neurons were infected at DIV 6–7 with lentiviruses encoding either KCTD12 shRNA or control shRNA and analyzed at DIV 13. KCTD12 shRNA significantly reduced the expression of endogenous KCTD12 protein as compared with control shRNA (55.8 ± 8.1% of control, KCTD12 protein was normalized to β-tubulin protein, p < 0.05; Fig. 6A). KCTD12 knockdown with shRNA resulted in a significant decrease of surface GABAB1 protein as compared with control shRNA (GABAB1a, 58.4 ± 9.7% of control, p < 0.01; GABAB1b, 66.4 ± 10.9%, p < 0.05; Fig. 6, A and B). In agreement with the results obtained from the cell surface biotinylation experiments, we found that knockdown of KCTD12 in hippocampal neurons significantly decreases the baclofen-induced K+ current density at the peak of the response (KCTD12 shRNA, 1.32 ± 0.20 pA/pF; control shRNA, 2.27 ± 0.21 pA/pF; p < 0.01; Fig. 6, C and D). Finally, we examined cell surface expression of GABAB receptors in cultured hippocampal neurons of Kctd12−/− mice (6). The cell surface biotinylation assay demonstrated that Kctd12−/− neurons express reduced amounts of GABAB1a and GABAB1b proteins at the plasma membrane in comparison with wild-type neurons (GABAB1a, 71.5 ± 9.6% of WT; GABAB1b, 77.9 ± 9.4% of WT, p < 0.05; Fig. 7, A and B). We additionally tested GABAB responses in Kctd12−/− and wild-type neurons. The GABAB receptor-mediated K+ current density at the peak of the response was slightly but significantly decreased in Kctd12−/− as compared with wild-type neurons (Kctd12−/−, 1.70 ± 0.07 pA/pF; WT, 1.94 ± 0.07 pA/pF; p < 0.05; Fig. 7, C and D). Based on these electrophysiological results, we conclude that the presence of KCTD12 increases the maximal GABAB receptor response in cultured hippocampal neurons.

Bottom Line: Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex.Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface.We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

Show MeSH
Related in: MedlinePlus