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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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KCTD12 stably interacts with GABAB receptors during receptor activation in transfected COS-1 cells.A, co-immunoprecipitation of KCTD12 with surface GABAB receptors from cells expressing Myc-GABAB1 (Myc-GB1), HA-GABAB2 (HA-GB2), and FLAG-KCTD12 (Flag-KCTD12) with and without receptor activation with baclofen (100 μm, 15 min). The amount of KCTD12 protein that co-immunoprecipitated with GABAB receptors was similar in the presence and absence of baclofen. The absence of α-actin in the immunoprecipitates (IP) supports that surface GABAB receptors were selectively isolated. Data are representative of 3 independent experiments done in duplicates. B, BRET donor saturation curves were generated in cells expressing fixed amounts of Myc-GABAB2, Rluc-KCTD12, or Rluc-KCTD10 and increasing amounts of GABAB1-YFP (GB1-YFP). Cells were preincubated with 100 μm baclofen for 15 min (filled circles and squares) or with PBS only (open circles and squares). BRET is expressed as milliBRET units (mBU) determined as net BRET × 1000. The results are the mean ± S.D. of 3–4 individual saturation experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).
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Figure 4: KCTD12 stably interacts with GABAB receptors during receptor activation in transfected COS-1 cells.A, co-immunoprecipitation of KCTD12 with surface GABAB receptors from cells expressing Myc-GABAB1 (Myc-GB1), HA-GABAB2 (HA-GB2), and FLAG-KCTD12 (Flag-KCTD12) with and without receptor activation with baclofen (100 μm, 15 min). The amount of KCTD12 protein that co-immunoprecipitated with GABAB receptors was similar in the presence and absence of baclofen. The absence of α-actin in the immunoprecipitates (IP) supports that surface GABAB receptors were selectively isolated. Data are representative of 3 independent experiments done in duplicates. B, BRET donor saturation curves were generated in cells expressing fixed amounts of Myc-GABAB2, Rluc-KCTD12, or Rluc-KCTD10 and increasing amounts of GABAB1-YFP (GB1-YFP). Cells were preincubated with 100 μm baclofen for 15 min (filled circles and squares) or with PBS only (open circles and squares). BRET is expressed as milliBRET units (mBU) determined as net BRET × 1000. The results are the mean ± S.D. of 3–4 individual saturation experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).

Mentions: Next we investigated whether the interaction of KCTD12 with GABAB receptors at the cell surface is regulated by receptor activity. We used anti-Myc antibodies to immunoprecipitate GABAB receptors from the cell surface of COS-1 cells expressing Myc-GABAB1, HA-GABAB2, and FLAG-KCTD12 (Fig. 4A). We co-immunoprecipitated equivalent amounts of KCTD12 with Myc-GABAB1 in the absence (PBS) and presence of baclofen (ratio of KCTD12:GABAB1 band intensity on Western blots; PBS, 0.84 ± 0.19; baclofen, 0.91 ± 0.11; p > 0.05, nonparametric Mann-Whitney test). These experiments show that KCTD12 is neither recruited nor dissociated from surface GABAB receptors upon receptor activation.


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)

KCTD12 stably interacts with GABAB receptors during receptor activation in transfected COS-1 cells.A, co-immunoprecipitation of KCTD12 with surface GABAB receptors from cells expressing Myc-GABAB1 (Myc-GB1), HA-GABAB2 (HA-GB2), and FLAG-KCTD12 (Flag-KCTD12) with and without receptor activation with baclofen (100 μm, 15 min). The amount of KCTD12 protein that co-immunoprecipitated with GABAB receptors was similar in the presence and absence of baclofen. The absence of α-actin in the immunoprecipitates (IP) supports that surface GABAB receptors were selectively isolated. Data are representative of 3 independent experiments done in duplicates. B, BRET donor saturation curves were generated in cells expressing fixed amounts of Myc-GABAB2, Rluc-KCTD12, or Rluc-KCTD10 and increasing amounts of GABAB1-YFP (GB1-YFP). Cells were preincubated with 100 μm baclofen for 15 min (filled circles and squares) or with PBS only (open circles and squares). BRET is expressed as milliBRET units (mBU) determined as net BRET × 1000. The results are the mean ± S.D. of 3–4 individual saturation experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).
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Related In: Results  -  Collection

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Figure 4: KCTD12 stably interacts with GABAB receptors during receptor activation in transfected COS-1 cells.A, co-immunoprecipitation of KCTD12 with surface GABAB receptors from cells expressing Myc-GABAB1 (Myc-GB1), HA-GABAB2 (HA-GB2), and FLAG-KCTD12 (Flag-KCTD12) with and without receptor activation with baclofen (100 μm, 15 min). The amount of KCTD12 protein that co-immunoprecipitated with GABAB receptors was similar in the presence and absence of baclofen. The absence of α-actin in the immunoprecipitates (IP) supports that surface GABAB receptors were selectively isolated. Data are representative of 3 independent experiments done in duplicates. B, BRET donor saturation curves were generated in cells expressing fixed amounts of Myc-GABAB2, Rluc-KCTD12, or Rluc-KCTD10 and increasing amounts of GABAB1-YFP (GB1-YFP). Cells were preincubated with 100 μm baclofen for 15 min (filled circles and squares) or with PBS only (open circles and squares). BRET is expressed as milliBRET units (mBU) determined as net BRET × 1000. The results are the mean ± S.D. of 3–4 individual saturation experiments. The curves were fitted using a nonlinear regression equation assuming a single binding site (GraphPad Prism).
Mentions: Next we investigated whether the interaction of KCTD12 with GABAB receptors at the cell surface is regulated by receptor activity. We used anti-Myc antibodies to immunoprecipitate GABAB receptors from the cell surface of COS-1 cells expressing Myc-GABAB1, HA-GABAB2, and FLAG-KCTD12 (Fig. 4A). We co-immunoprecipitated equivalent amounts of KCTD12 with Myc-GABAB1 in the absence (PBS) and presence of baclofen (ratio of KCTD12:GABAB1 band intensity on Western blots; PBS, 0.84 ± 0.19; baclofen, 0.91 ± 0.11; p > 0.05, nonparametric Mann-Whitney test). These experiments show that KCTD12 is neither recruited nor dissociated from surface GABAB receptors upon receptor activation.

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