Limits...
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.

Show MeSH

Related in: MedlinePlus

KCTD12 assembles with GABAB receptors at the cytoplasmic side of the ER membrane in transfected COS-1 cells.A, cells co-expressing combinations of FLAG-KCTD12 and Myc-GABAB2 (GB2) were metabolically labeled with [35S]methionine for 15 min. After immunoprecipitation (IP) of FLAG-KCTD12, the precipitated 35S-labeled proteins were separated by SDS-PAGE and visualized using autoradiography. 35S-labeled Myc-GB2 and FLAG-KCTD12 proteins were identified by cross-correlation of autoradiographs with Western blots developed with anti-Myc and anti-FLAG antibodies, respectively. 35S-labeled Myc-GB2 protein co-precipitates with FLAG-KCTD12 protein, showing that KCTD12 interacts with newly synthesized GB2 residing in the ER. MW, molecular weight. B, a YFP protein fragment complementation assay between HA-GB2-YFP1 and FLAG-KCTD12-YFP2 was used to visualize the GABAB2-KCTD12 interaction in the secretory pathway. The fluorescence of reconstituted YFP overlapped with the ER marker calnexin, which was detected with anti-calnexin antibodies. As a negative control, expression of HA-GB2Y902A-YFP1, which does not bind to KCTD12, together with FLAG-KCTD12-YFP2 did not reconstitute YFP. Anti-HA antibodies were used to identify HA-GB2-YFP1 and HA-GB2Y902A-YFP1 in transfected cells (scale bar, 20 μm). C, Endo H and N-glycosidase F (PNGase F) treatment of Myc-GABAB1 (GB1) and Myc-GABAB1ASAA (GB1ASAA), a mutant GB1 protein that escapes ER retention. In contrast to GB1, the GB1ASAA protein is partially Endo H-resistant (filled arrow). D, glycosylation patterns of cell lysates expressing combinations of Myc-GABAB1 (GB1), HA-GABAB2 (GB2), and FLAG-KCTD12 (KCTD12) were analyzed on Western blots following Endo H or N-glycosidase F treatment. Endo H-resistant (filled arrows) and -sensitive (open arrows) forms of GB1 and GB2 are indicated. E, quantification of Endo H-resistant GB1 and GB2 protein did not reveal any significant alterations of subunit maturation in the presence of KCTD12. Data are the means ± S.E. of 3 independent experiments done in duplicates, p > 0.05. ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750179&req=5

Figure 3: KCTD12 assembles with GABAB receptors at the cytoplasmic side of the ER membrane in transfected COS-1 cells.A, cells co-expressing combinations of FLAG-KCTD12 and Myc-GABAB2 (GB2) were metabolically labeled with [35S]methionine for 15 min. After immunoprecipitation (IP) of FLAG-KCTD12, the precipitated 35S-labeled proteins were separated by SDS-PAGE and visualized using autoradiography. 35S-labeled Myc-GB2 and FLAG-KCTD12 proteins were identified by cross-correlation of autoradiographs with Western blots developed with anti-Myc and anti-FLAG antibodies, respectively. 35S-labeled Myc-GB2 protein co-precipitates with FLAG-KCTD12 protein, showing that KCTD12 interacts with newly synthesized GB2 residing in the ER. MW, molecular weight. B, a YFP protein fragment complementation assay between HA-GB2-YFP1 and FLAG-KCTD12-YFP2 was used to visualize the GABAB2-KCTD12 interaction in the secretory pathway. The fluorescence of reconstituted YFP overlapped with the ER marker calnexin, which was detected with anti-calnexin antibodies. As a negative control, expression of HA-GB2Y902A-YFP1, which does not bind to KCTD12, together with FLAG-KCTD12-YFP2 did not reconstitute YFP. Anti-HA antibodies were used to identify HA-GB2-YFP1 and HA-GB2Y902A-YFP1 in transfected cells (scale bar, 20 μm). C, Endo H and N-glycosidase F (PNGase F) treatment of Myc-GABAB1 (GB1) and Myc-GABAB1ASAA (GB1ASAA), a mutant GB1 protein that escapes ER retention. In contrast to GB1, the GB1ASAA protein is partially Endo H-resistant (filled arrow). D, glycosylation patterns of cell lysates expressing combinations of Myc-GABAB1 (GB1), HA-GABAB2 (GB2), and FLAG-KCTD12 (KCTD12) were analyzed on Western blots following Endo H or N-glycosidase F treatment. Endo H-resistant (filled arrows) and -sensitive (open arrows) forms of GB1 and GB2 are indicated. E, quantification of Endo H-resistant GB1 and GB2 protein did not reveal any significant alterations of subunit maturation in the presence of KCTD12. Data are the means ± S.E. of 3 independent experiments done in duplicates, p > 0.05. ns, not significant.

Mentions: We next determined where in the biosynthetic pathway KCTD12 assembles with GABAB receptors. We pulse-labeled COS-1 cells expressing GABAB2 and KCTD12 for 15 min with [35S]methionine, at which time newly synthesized transmembrane proteins have not yet progressed beyond the ER (27, 28). After 15 min of biosynthesis, immunoprecipitation with anti-FLAG antibodies not only purifies FLAG-KCTD12 but also efficiently co-precipitates 35S-labeled Myc-GABAB2 (Fig. 3A). This shows that FLAG-KCTD12 binds to newly synthesized 35S-labeled Myc-GABAB2 residing in the ER. The C termini of the GABAB1 and GABAB2 proteins are expected to face the cytoplasmic side of the ER membrane (7). Therefore KCTD12, a cytosolic protein, is assumed to assemble with GABAB2 at the cytoplasmic side of the ER membrane. We additionally studied assembly of KCTD12 with GABAB receptors in living cells using a YFP protein fragment complementation assay (15). We fused the YFP1 and YFP2 fragments to the C termini of GABAB2 (GABAB2-YFP1) and KCTD12 (KCTD12-YFP2), respectively. Co-expression of GABAB2-YFP1 with KCTD12-YFP2 in COS-1 cells produced YFP fluorescence due to reconstitution of a functional YFP protein (Fig. 3B). The YFP fluorescence fully overlapped with the immunostaining pattern for the ER marker protein calnexin, showing that YFP reconstitution already takes place at the level of the ER (Fig. 3B). Binding of KCTD12-YFP2 to GABAB2-YFP1 was further confirmed in co-immunoprecipitation experiments (data not shown). In control experiments, we did not detect YFP fluorescence when expressing GABAB2-YFP1 or KCTD12-YFP2 in the absence of each other (data not shown). This indicates that the YFP fragments do not generate fluorescence on their own. Moreover, we did not observe YFP reconstitution when expressing GABAB2Y902A-YFP1, which does not bind to KCTD12, together with KCTD12-YFP2. Altogether, our data support that KCTD12 first assembles with GABAB receptors at the cytoplasmic side of the ER membrane.


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 assembles with GABAB receptors at the cytoplasmic side of the ER membrane in transfected COS-1 cells.A, cells co-expressing combinations of FLAG-KCTD12 and Myc-GABAB2 (GB2) were metabolically labeled with [35S]methionine for 15 min. After immunoprecipitation (IP) of FLAG-KCTD12, the precipitated 35S-labeled proteins were separated by SDS-PAGE and visualized using autoradiography. 35S-labeled Myc-GB2 and FLAG-KCTD12 proteins were identified by cross-correlation of autoradiographs with Western blots developed with anti-Myc and anti-FLAG antibodies, respectively. 35S-labeled Myc-GB2 protein co-precipitates with FLAG-KCTD12 protein, showing that KCTD12 interacts with newly synthesized GB2 residing in the ER. MW, molecular weight. B, a YFP protein fragment complementation assay between HA-GB2-YFP1 and FLAG-KCTD12-YFP2 was used to visualize the GABAB2-KCTD12 interaction in the secretory pathway. The fluorescence of reconstituted YFP overlapped with the ER marker calnexin, which was detected with anti-calnexin antibodies. As a negative control, expression of HA-GB2Y902A-YFP1, which does not bind to KCTD12, together with FLAG-KCTD12-YFP2 did not reconstitute YFP. Anti-HA antibodies were used to identify HA-GB2-YFP1 and HA-GB2Y902A-YFP1 in transfected cells (scale bar, 20 μm). C, Endo H and N-glycosidase F (PNGase F) treatment of Myc-GABAB1 (GB1) and Myc-GABAB1ASAA (GB1ASAA), a mutant GB1 protein that escapes ER retention. In contrast to GB1, the GB1ASAA protein is partially Endo H-resistant (filled arrow). D, glycosylation patterns of cell lysates expressing combinations of Myc-GABAB1 (GB1), HA-GABAB2 (GB2), and FLAG-KCTD12 (KCTD12) were analyzed on Western blots following Endo H or N-glycosidase F treatment. Endo H-resistant (filled arrows) and -sensitive (open arrows) forms of GB1 and GB2 are indicated. E, quantification of Endo H-resistant GB1 and GB2 protein did not reveal any significant alterations of subunit maturation in the presence of KCTD12. Data are the means ± S.E. of 3 independent experiments done in duplicates, p > 0.05. ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750179&req=5

Figure 3: KCTD12 assembles with GABAB receptors at the cytoplasmic side of the ER membrane in transfected COS-1 cells.A, cells co-expressing combinations of FLAG-KCTD12 and Myc-GABAB2 (GB2) were metabolically labeled with [35S]methionine for 15 min. After immunoprecipitation (IP) of FLAG-KCTD12, the precipitated 35S-labeled proteins were separated by SDS-PAGE and visualized using autoradiography. 35S-labeled Myc-GB2 and FLAG-KCTD12 proteins were identified by cross-correlation of autoradiographs with Western blots developed with anti-Myc and anti-FLAG antibodies, respectively. 35S-labeled Myc-GB2 protein co-precipitates with FLAG-KCTD12 protein, showing that KCTD12 interacts with newly synthesized GB2 residing in the ER. MW, molecular weight. B, a YFP protein fragment complementation assay between HA-GB2-YFP1 and FLAG-KCTD12-YFP2 was used to visualize the GABAB2-KCTD12 interaction in the secretory pathway. The fluorescence of reconstituted YFP overlapped with the ER marker calnexin, which was detected with anti-calnexin antibodies. As a negative control, expression of HA-GB2Y902A-YFP1, which does not bind to KCTD12, together with FLAG-KCTD12-YFP2 did not reconstitute YFP. Anti-HA antibodies were used to identify HA-GB2-YFP1 and HA-GB2Y902A-YFP1 in transfected cells (scale bar, 20 μm). C, Endo H and N-glycosidase F (PNGase F) treatment of Myc-GABAB1 (GB1) and Myc-GABAB1ASAA (GB1ASAA), a mutant GB1 protein that escapes ER retention. In contrast to GB1, the GB1ASAA protein is partially Endo H-resistant (filled arrow). D, glycosylation patterns of cell lysates expressing combinations of Myc-GABAB1 (GB1), HA-GABAB2 (GB2), and FLAG-KCTD12 (KCTD12) were analyzed on Western blots following Endo H or N-glycosidase F treatment. Endo H-resistant (filled arrows) and -sensitive (open arrows) forms of GB1 and GB2 are indicated. E, quantification of Endo H-resistant GB1 and GB2 protein did not reveal any significant alterations of subunit maturation in the presence of KCTD12. Data are the means ± S.E. of 3 independent experiments done in duplicates, p > 0.05. ns, not significant.
Mentions: We next determined where in the biosynthetic pathway KCTD12 assembles with GABAB receptors. We pulse-labeled COS-1 cells expressing GABAB2 and KCTD12 for 15 min with [35S]methionine, at which time newly synthesized transmembrane proteins have not yet progressed beyond the ER (27, 28). After 15 min of biosynthesis, immunoprecipitation with anti-FLAG antibodies not only purifies FLAG-KCTD12 but also efficiently co-precipitates 35S-labeled Myc-GABAB2 (Fig. 3A). This shows that FLAG-KCTD12 binds to newly synthesized 35S-labeled Myc-GABAB2 residing in the ER. The C termini of the GABAB1 and GABAB2 proteins are expected to face the cytoplasmic side of the ER membrane (7). Therefore KCTD12, a cytosolic protein, is assumed to assemble with GABAB2 at the cytoplasmic side of the ER membrane. We additionally studied assembly of KCTD12 with GABAB receptors in living cells using a YFP protein fragment complementation assay (15). We fused the YFP1 and YFP2 fragments to the C termini of GABAB2 (GABAB2-YFP1) and KCTD12 (KCTD12-YFP2), respectively. Co-expression of GABAB2-YFP1 with KCTD12-YFP2 in COS-1 cells produced YFP fluorescence due to reconstitution of a functional YFP protein (Fig. 3B). The YFP fluorescence fully overlapped with the immunostaining pattern for the ER marker protein calnexin, showing that YFP reconstitution already takes place at the level of the ER (Fig. 3B). Binding of KCTD12-YFP2 to GABAB2-YFP1 was further confirmed in co-immunoprecipitation experiments (data not shown). In control experiments, we did not detect YFP fluorescence when expressing GABAB2-YFP1 or KCTD12-YFP2 in the absence of each other (data not shown). This indicates that the YFP fragments do not generate fluorescence on their own. Moreover, we did not observe YFP reconstitution when expressing GABAB2Y902A-YFP1, which does not bind to KCTD12, together with KCTD12-YFP2. Altogether, our data support that KCTD12 first assembles with GABAB receptors at the cytoplasmic side of the ER membrane.

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