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. 2001 Feb 15;21(4):1203-10.
doi: 10.1523/JNEUROSCI.21-04-01203.2001.

The C-terminal Domains of the GABA(b) Receptor Subunits Mediate Intracellular Trafficking but Are Not Required for Receptor Signaling

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Free PMC article

The C-terminal Domains of the GABA(b) Receptor Subunits Mediate Intracellular Trafficking but Are Not Required for Receptor Signaling

A R Calver et al. J Neurosci. .
Free PMC article

Abstract

GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

Figures

Fig. 1.
Fig. 1.
GABAB receptor subunit truncations and chimeras used in transfection experiments. c-myc and HA are epitope tags for immunocytochemistry and Western blotting. TMD1–7 are the seven transmembrane domains; coiled-coil is the C-terminal domain implicated in the interaction between GABAB1b and GABAB2.
Fig. 2.
Fig. 2.
Intracellular retention of GABAB1b is mediated by the coiled-coil motif within the C terminal of the protein.A–H, HEK-293 cells were transiently transfected with constructs encoding either the full-length GABAB1b(A, B) or progressive deletions of GABAB1b(C–H). All of the full-length and truncated subunits were tagged with a c-myc epitope recognized by the 9E10 antibody. Transfected cells were examined after 24 hr by immunofluorescence using the 9E10 antibody either with (B, D, F, H) or without (A, C, E, G) permeabilization. GABAB1b and GABAB1bΔ806 are both retained intracellularly (A–D), whereas GABAB1bΔ771 and GABAB1bΔ747 are both expressed on the cell surface (E–H).Insets, Diagrams of the truncations are shown, with the coiled-coil region necessary for receptor dimerization indicated inred. All of the transfections were performed and analyzed at least three times, and the results with each construct were consistent. Scale bar, 10 μm.
Fig. 3.
Fig. 3.
The C-terminal domain of GABAB1bis sufficient to sequester GABAB2 within the cell.A–H, HEK-293 cells were transfected with constructs encoding either the full-length GABAB2 (A, B), GABAB2Δ748 (C, D), GABAB2/1bC (E, F), or GABAB2/1bC + GABAB2 (G, H). All of the full-length and truncated subunits were tagged with an HA epitope recognized by the 3F10 antibody, except for the full-length GABAB2 used in the cotransfection (G, H) that was not epitope-tagged. Transfected cells were examined after 24 hr by immunofluorescence using the 3F10 antibody either with (B, D, F, H) or without (A, C, E, G) permeabilization. Both full-length GABAB2 and GABAB2Δ748 when transfected alone are expressed on the cell surface (A–D), whereas GABAB2/1bC is retained within the cell (E, F). The HA-tagged GABAB2/1bC is able to reach the cell surface and be detected by anti-HA, however, when coexpressed with the untagged GABAB2 (G, H). Insets, Diagrams of the truncations and chimeras are shown; GABAB2 sequences are shown inred, whereas GABAB1b sequences are shown inblack. All of the transfections were performed and analyzed at least three times, and the results with each construct were consistent. Scale bar, 10 μm.
Fig. 4.
Fig. 4.
Removal of the C terminal of GABAB1bdoes not affect ligand binding. Cells transiently transfected with either GABAB1b alone, GABAB1b + GABAB2, GABAB1bΔ747 alone, or GABAB1bΔ747 + GABAB2 specifically bound [3H]CGP-54626, and this could be completely displaced by GABA (10 mm), with pKi values of 3.60 ± 0.13, 4.14 ± 0.05, 3.70 ± 0.10, and 4.00 ± 0.09, respectively. Data are expressed as means ± SEM (n = 4–6). All receptor subunits were epitope-tagged as described in Materials and Methods.
Fig. 5.
Fig. 5.
GABAB1b subunits lacking the intracellular C terminal are nonfunctional, whereas heterodimers formed between the C-terminally truncated GABAB1 and either full-length or C-terminally truncated GABAB2 signal via G-proteins. HEK-293 cells were cotransfected with the expression constructs shown together with the chimeric G-protein Gqi5 and assayed for intracellular Ca2+ mobilization in response to GABA stimulation in a FLIPR. A, No response was observed from mock-transfected cells or from cells transfected with GABAB1b or GABAB1bΔ747 on their own, whereas a robust functional response was seen when GABAB1b was cotransfected with GABAB2(pEC50 = 7.08 ± 0.02). B, A similar response was seen when GABAB2 was cotransfected with GABAB1bΔ747 or when GABAB2 was cotransfected with GABAB1b [pEC50(GABAB1b + GABAB2) = 7.08 ± 0.02; pEC50 (GABAB1bΔ747 + GABAB2) = 6.93 ± 0.05].C, A functional GABAB receptor was also detected in FLIPR when GABAB1bΔ747 was cotransfected with GABAB2Δ748 and Gqi5 (pEC50= 6.34 ± 0.01). The data in A–C are taken from a single representative experiment. All receptor subunits were epitope-tagged as described in Materials and Methods.
Fig. 6.
Fig. 6.
Heterodimers between GABAB1b and GABAB2 form in the absence of the C-terminal coiled-coil interaction. A, Western blot to show specificity of anti-GABAB1b antibody. Single bands are observed in lanes containing either rat brain membranes (lane 3) or cells transfected with GABAB1b(lane 2); no bands are observed in untransfected cells (lane 1). B, Immunoprecipitation from transfected cell membranes with anti-GABAB1b antibody followed by Western blotting with anti-GABAB2. HEK-293 cell membranes were prepared from mock-transfected cells (lane 1) and from cells transiently transfected with GABAB1b + GABAB2 (lane 2), GABAB1bΔ748 (lane 3), GABAB2(lane 4), or GABAB1bΔ748 + GABAB2 (lane 5). This experiment clearly demonstrates an interaction between GABAB1bΔ748 and GABAB2 (lane 5), in addition to the strong interaction between GABAB1b and GABAB2(lane 2). Numbers on the leftindicate the position of molecular weight markers, expressed in kilodaltons.

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