The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling

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.

Fig. 1.

GABA_B 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 GABA_B1b and GABA_B2.

Fig. 2.

Intracellular retention of GABA_B1b 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 GABA_B1b(A, B) or progressive deletions of GABA_B1b(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. GABA_B1b and GABA_B1bΔ806 are both retained intracellularly (A–D), whereas GABA_B1bΔ771 and GABA_B1bΔ747 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.

The C-terminal domain of GABA_B1bis sufficient to sequester GABA_B2 within the cell.A–H, HEK-293 cells were transfected with constructs encoding either the full-length GABA_B2 (A, B), GABA_B2Δ748 (C, D), GABA_B2/1bC (E, F), or GABA_B2/1bC + GABA_B2 (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 GABA_B2 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 GABA_B2 and GABA_B2Δ748 when transfected alone are expressed on the cell surface (A–D), whereas GABA_B2/1bC is retained within the cell (E, F). The HA-tagged GABA_B2/1bC is able to reach the cell surface and be detected by anti-HA, however, when coexpressed with the untagged GABA_B2 (G, H). Insets, Diagrams of the truncations and chimeras are shown; GABA_B2 sequences are shown inred, whereas GABA_B1b 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.

Removal of the C terminal of GABA_B1bdoes not affect ligand binding. Cells transiently transfected with either GABA_B1b alone, GABA_B1b + GABA_B2, GABA_B1bΔ747 alone, or GABA_B1bΔ747 + GABA_B2 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.

GABA_B1b subunits lacking the intracellular C terminal are nonfunctional, whereas heterodimers formed between the C-terminally truncated GABA_B1 and either full-length or C-terminally truncated GABA_B2 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 GABA_B1b or GABA_B1bΔ747 on their own, whereas a robust functional response was seen when GABA_B1b was cotransfected with GABA_B2(pEC₅₀ = 7.08 ± 0.02). B, A similar response was seen when GABA_B2 was cotransfected with GABA_B1bΔ747 or when GABA_B2 was cotransfected with GABA_B1b [pEC₅₀(GABA_B1b + GABA_B2) = 7.08 ± 0.02; pEC₅₀ (GABA_B1bΔ747 + GABA_B2) = 6.93 ± 0.05].C, A functional GABA_B receptor was also detected in FLIPR when GABA_B1bΔ747 was cotransfected with GABA_B2Δ748 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.

Heterodimers between GABA_B1b and GABA_B2 form in the absence of the C-terminal coiled-coil interaction. A, Western blot to show specificity of anti-GABA_B1b antibody. Single bands are observed in lanes containing either rat brain membranes (lane 3) or cells transfected with GABA_B1b(lane 2); no bands are observed in untransfected cells (lane 1). B, Immunoprecipitation from transfected cell membranes with anti-GABA_B1b antibody followed by Western blotting with anti-GABA_B2. HEK-293 cell membranes were prepared from mock-transfected cells (lane 1) and from cells transiently transfected with GABA_B1b + GABA_B2 (lane 2), GABA_B1bΔ748 (lane 3), GABA_B2(lane 4), or GABA_B1bΔ748 + GABA_B2 (lane 5). This experiment clearly demonstrates an interaction between GABA_B1bΔ748 and GABA_B2 (lane 5), in addition to the strong interaction between GABA_B1b and GABA_B2(lane 2). Numbers on the leftindicate the position of molecular weight markers, expressed in kilodaltons.