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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 reduces constitutive GABAB receptor internalization in transfected COS-1 cells.A, GABAB receptor internalization in the presence or absence of KCTD12 was investigated using a biotinylation assay. Cell were biotinylated on ice for 15 min, washed, and then either incubated on ice for 120 min to prevent internalization of cell surface proteins (Total surface) or incubated at 37 °C for 120 min to allow internalization (Internalized). Biotinylated total surface and internalized proteins were purified with NeutrAvidin-Sepharose. GABAB1 (GB1) and KCTD12 protein was revealed on Western blots. For purification of biotinylated internalized protein, the biotin label at the cell surface was first removed by incubation with reduced glutathione (GSH). Biotinylated total surface protein cleaved by GSH (GSH cleavage) demonstrates that GSH efficiently removes the biotin label from surface proteins. Internalized GB1 and KCTD12 proteins (Internalized) were always analyzed in duplicates. B, quantification of GABAB receptor internalization in the absence and presence of KCTD12. The percentage of the total GB1 protein that was internalized was calculated after subtraction of uncleaved GB1 protein after GSH cleavage at the cell surface. Bars are the means ± S.E. of 3 separate experiments. *, p < 0.05.
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Figure 5: KCTD12 reduces constitutive GABAB receptor internalization in transfected COS-1 cells.A, GABAB receptor internalization in the presence or absence of KCTD12 was investigated using a biotinylation assay. Cell were biotinylated on ice for 15 min, washed, and then either incubated on ice for 120 min to prevent internalization of cell surface proteins (Total surface) or incubated at 37 °C for 120 min to allow internalization (Internalized). Biotinylated total surface and internalized proteins were purified with NeutrAvidin-Sepharose. GABAB1 (GB1) and KCTD12 protein was revealed on Western blots. For purification of biotinylated internalized protein, the biotin label at the cell surface was first removed by incubation with reduced glutathione (GSH). Biotinylated total surface protein cleaved by GSH (GSH cleavage) demonstrates that GSH efficiently removes the biotin label from surface proteins. Internalized GB1 and KCTD12 proteins (Internalized) were always analyzed in duplicates. B, quantification of GABAB receptor internalization in the absence and presence of KCTD12. The percentage of the total GB1 protein that was internalized was calculated after subtraction of uncleaved GB1 protein after GSH cleavage at the cell surface. Bars are the means ± S.E. of 3 separate experiments. *, p < 0.05.

Mentions: Because we did not observe significant changes in GABAB receptor maturation and forward trafficking in the presence of KCTD12 (see above), we addressed whether KCTD12 possibly increases receptor surface levels by reducing constitutive endocytosis (20, 26, 33, 34). A cell surface biotinylation assay (20) indeed reveals a significantly reduced GABAB receptor endocytosis in the presence of KCTD12 (Fig. 5A). Quantitative analysis showed that the percentage of total GABAB1 protein internalized within 120 min was significantly smaller in the presence of KCTD12 (KCTD12, 16 ± 5%; without KCTD12, 33 ± 6%; p < 0.05, nonparametric Mann-Whitney test; Fig. 5B). The rate of constitutive GABAB receptor internalization in the absence of KCTD12 was similar to the rate reported in an earlier study (20). We conclude that KCTD12 increases cell surface GABAB receptor expression by reducing constitutive receptor internalization. Co-immunoprecipitation experiments further reveal that GABAB1, GABAB2, and KCTD12 endocytose in associate with each other as a protein complex (Fig. 5A).


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 reduces constitutive GABAB receptor internalization in transfected COS-1 cells.A, GABAB receptor internalization in the presence or absence of KCTD12 was investigated using a biotinylation assay. Cell were biotinylated on ice for 15 min, washed, and then either incubated on ice for 120 min to prevent internalization of cell surface proteins (Total surface) or incubated at 37 °C for 120 min to allow internalization (Internalized). Biotinylated total surface and internalized proteins were purified with NeutrAvidin-Sepharose. GABAB1 (GB1) and KCTD12 protein was revealed on Western blots. For purification of biotinylated internalized protein, the biotin label at the cell surface was first removed by incubation with reduced glutathione (GSH). Biotinylated total surface protein cleaved by GSH (GSH cleavage) demonstrates that GSH efficiently removes the biotin label from surface proteins. Internalized GB1 and KCTD12 proteins (Internalized) were always analyzed in duplicates. B, quantification of GABAB receptor internalization in the absence and presence of KCTD12. The percentage of the total GB1 protein that was internalized was calculated after subtraction of uncleaved GB1 protein after GSH cleavage at the cell surface. Bars are the means ± S.E. of 3 separate experiments. *, p < 0.05.
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Figure 5: KCTD12 reduces constitutive GABAB receptor internalization in transfected COS-1 cells.A, GABAB receptor internalization in the presence or absence of KCTD12 was investigated using a biotinylation assay. Cell were biotinylated on ice for 15 min, washed, and then either incubated on ice for 120 min to prevent internalization of cell surface proteins (Total surface) or incubated at 37 °C for 120 min to allow internalization (Internalized). Biotinylated total surface and internalized proteins were purified with NeutrAvidin-Sepharose. GABAB1 (GB1) and KCTD12 protein was revealed on Western blots. For purification of biotinylated internalized protein, the biotin label at the cell surface was first removed by incubation with reduced glutathione (GSH). Biotinylated total surface protein cleaved by GSH (GSH cleavage) demonstrates that GSH efficiently removes the biotin label from surface proteins. Internalized GB1 and KCTD12 proteins (Internalized) were always analyzed in duplicates. B, quantification of GABAB receptor internalization in the absence and presence of KCTD12. The percentage of the total GB1 protein that was internalized was calculated after subtraction of uncleaved GB1 protein after GSH cleavage at the cell surface. Bars are the means ± S.E. of 3 separate experiments. *, p < 0.05.
Mentions: Because we did not observe significant changes in GABAB receptor maturation and forward trafficking in the presence of KCTD12 (see above), we addressed whether KCTD12 possibly increases receptor surface levels by reducing constitutive endocytosis (20, 26, 33, 34). A cell surface biotinylation assay (20) indeed reveals a significantly reduced GABAB receptor endocytosis in the presence of KCTD12 (Fig. 5A). Quantitative analysis showed that the percentage of total GABAB1 protein internalized within 120 min was significantly smaller in the presence of KCTD12 (KCTD12, 16 ± 5%; without KCTD12, 33 ± 6%; p < 0.05, nonparametric Mann-Whitney test; Fig. 5B). The rate of constitutive GABAB receptor internalization in the absence of KCTD12 was similar to the rate reported in an earlier study (20). We conclude that KCTD12 increases cell surface GABAB receptor expression by reducing constitutive receptor internalization. Co-immunoprecipitation experiments further reveal that GABAB1, GABAB2, and KCTD12 endocytose in associate with each other as a protein complex (Fig. 5A).

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