GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer

Abstract

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

Fig. 1.

Intracellular loop sequence alignments and summary of GABA_B1 and GABA_B2 constructs.a, Alignment of the second and third intracellular loops of GABA_B1 (GABAB1) and GABA_B2(GABAB2) subunits with members of the mGluR family. The alignment was produced in Alscript using HMMALIGN and the G-protein-coupled receptors database (GPCRDB) (Barton, 1993; Horn et al., 1998). The shading indicates different levels of amino acid conservation, in which residues are colored if eight or more residues show conservation of amino acid properties: blue shading, small; yellow shading, hydrophobic; red characters, polar. Transmembrane helices (TMs, dark blue) are displayed for mGluR1 (as assigned in SwissProt, entry MGR1 HUMAN).Boxed letters indicate residues that are negatively charged. Conserved residues discussed in Results are shaded ingray. The alignment has been extracted from a larger alignment of family 3 GPCRs taken from GPCRDB (Horn et al., 1998).b, Enlarged alignment of the second intracellular loop of both GABA_B1 and GABA_B2 subunits. Bold red letters indicate residues mutated in this study.c, GABA_B receptor subunit truncations and chimeras. Epitope tags are marked as red (c-myc) orblue (HA) boxes. Transmembrane domains 1–7 (TMD 1–7) are represented by black striped boxes.. The coiled-coil domains are depicted bygreen striped boxes.

Fig. 2.

Residues within the second intracellular loop of GABA_B2 are critical for agonist-induced GABA_B receptor signaling. a, Schematic representation of GABA_B2 il2. Arrowsindicate residues mutated. b, HEK293 cells were transiently transfected with wtGABA_B1 (GB1) and wtGABA_B2 (GB2), GB2^K586E, GB2^K590E, GB2^K586E/M587E, or GB2^tripleE_. Cell surface expression of GABA_B subunits was examined by immunofluorescence using an anti-myc antibody for GABA_B1 (i–v) and an anti-HA antibody for GABA_B2 (vi–x).c, Representative FLIPR analysis of intracellular Ca²⁺ changes after GABA stimulation of cells transiently transfected with G_qi5, wtGABA_B1 (GB1), and wtGABA_B2(GB2) GB2^K586E, GB2^K590E, GB2^K586E/M587E, or GB2^tripleE. Expression of single and double GABA_B2 mutants resulted in decreased functional responses compared with wtGABA_B2, with the GABA_B2^tripleE mutant showing no response at all. d, Representative FLIPR analysis of intracellular Ca²⁺ changes after GABA stimulation of cells transiently transfected with G_qi5, wtGABA_B1 (GB1), and wtGABA_B2(GB2), GB2^K586A, GB2^K590A, GB2^K586A/M587A, or GB2^tripleA. Single and double GABA_B2 mutants resulted in decreased functional responses compared with wtGABA_B2, with the GABA_B2^tripleA mutant completely unresponsive to GABA. e, Mutation of residues within the second intracellular loop of GABA_B2 do not affect agonist or antagonist ligand binding. Membrane homogenates prepared from cells transiently transfected with wtGABA_B1(GB1) and wtGABA_B2(GB2), GB2^K586E, GB2^K590E, GB2^K586E/M587E, or GB2^tripleE specifically bound [³H]CGP54626. This could be completely displaced by GABA (10 mm). Data are expressed as means ± SEM (n = 3).

Fig. 3.

Acidic residues within the second intracellular loop of GABA_B1 are not critical for agonist-induced GABA_B receptor signaling. a, Schematic representation of GABA_B1 il2.Arrows indicate residues mutated. b, HEK293 cells were transiently transfected with wtGABA_B1(GB1), GB1^E579K, GB1^E583K, GB1^E579K/E580K, GB1^tripleK, or GB1_C-^tripleK alone or with wtGABA_B2. Cell surface expression of GABA_B1subunits was examined by immunofluorescence using an anti-myc antibody in the absence (i, iii,iv) or presence (ii, v) of a permeabilizing detergent. GABA_B1 is only found at the cell surface when expressed with GABA_B2, as shown with the GABA_B1^tripleK construct (i–iii). The C-terminally truncated GABA_B1is expressed at the cell surface in the absence of GABA_B2, as shown with the GABA_B1C-^tripleK mutant (iv, v). c, Representative FLIPR analysis of cells transiently transfected with G_qi5, wtGABA_B2 (GB2), and wtGABA_B1 (GB1), GB1^E579K, GB1^E583K, GB1^E579K/E580K, or GB1^tripleK. No differences in functional GABA responses were observed between mutant and wild-type GABA_B1subunits. d, Representative FLIPR analysis of cells transiently transfected with G_qi5 and GB1_C-^tripleK. Expression of GB1_C-^tripleK alone did not give a functional response.

Fig. 4.

Basic residues within the second intracellular loop of GABA_B1 are not critical for agonist-induced GABA_B receptor signaling. a, Schematic representation of GABA_B1 il2.Arrows indicate residues mutated. b, HEK293 cells were transiently transfected with wtGABA_B2(GB2) and wtGABA_B1 (GB1), GB1^K577/578A, GB1^K581/582A, GB1^KQA, or GB1^KQE_. GABA_B1 is only found at the cell surface when expressed with GABA_B2, as shown by immunofluorescence using an anti-myc antibody in the absence (i–v) of a permeabilizing detergent. c, Representative FLIPR analysis of cells transiently transfected with G_qi5, wtGABA_B2 (GB2), and wtGABA_B1 (GB1)_,GB1^K577/578A, GB1^K581/582A, GB1^KQA, or GB1^KQE. Both mutant and wild-type GABA_B1 subunits are functional when coexpressed with wtGABA_B2.

Fig. 5.

Loss of functional coupling after exchange of GABA_B1 and GABA_B2 second intracellular loop charged residues. a, HEK293 cells were transiently transfected with GABA_B1^tripleK(GB1^tripleK) and GABA_B2^tripleE(GB2^tripleE). Cell surface expression of GABA_B subunits was examined by immunofluorescence using an anti-myc antibody for GB1^tripleK (i) and an anti-HA antibody for GB2^tripleE (ii).b, Representative FLIPR analysis showing no functional response to GABA in cells transiently transfected with G_qi5, GB1^tripleK, and GB2^tripleE.

Fig. 6.

Coupling of wtGABA_B2 and GABA_B2 mutants to N-type Ca²⁺ channels in sympathetic neurons. a, Cell surface expression of wild-type and mutant GABA_B2 subunits examined by immunofluorescence using an anti-HA antibody. b, Currents were recorded by stepping for 100 msec every 20 sec from −90 to 0 mV and leak-corrected by subtracting currents remaining after substituting 5 mm Co²⁺ for Ba²⁺. Records show superimposed leak-subtracted currents in the absence (black line) and presence (red line) of 50 μm baclofen for neurons injected 16–24 hr before recordings with 100–150 ng/μl wtGABA_B2 (GB2), GB2^K590E, or GB2^tripleE. c, Bar charts show the mean inhibition of I_Ba amplitude by 50 μm baclofen, 10 msec after voltage stepping, in neurons injected with GB2, GB2^K590E, or GB2^tripleE. Error bars show SEM; nindicates number of cells. Note that GABA_B2^K590E still mediates inhibition of Ca²⁺ channel current, whereas GABA_B2^tripleE does not couple to Ca²⁺ channels. d, Plots show concentration dependence of Ca²⁺ current inhibition by baclofen in SCG neurons injected with GB2 (n = 5) or GB2^K590E (n = 3). Curves were fitted to pooled data points (mean ± SEM) using Origin 5 software to the Hill equation y =y_{max [infi]} ·x^nH/ (x^nH+ K^nH), where y is observed percentage of inhibition, y_max is the extrapolated maximal percentage of inhibition, x is baclofen concentration (micromolar), K is IC₅₀ (micromolar), and nH is the Hill coefficient. For GABA_B2^K590E, IC₅₀ of 1.04 ± 0.19 μm; nH of 1.23 ± 0.24; percentage of maximal inhibition, 42.03 ± 1.92%. For GABA_B2, IC50 of 0.38 ± 0.06 μm;nH of 1.21 ± 0.19; percentage of maximal inhibition, 52.55 ± 2.02%. Note that GABA_B2^K590Einhibits Ca²⁺ current less effectively than GABA_B2 and that plots could not be made for GABA_B2^tripleE because there was no detectable functional response.

Fig. 7.

GABA_B2 expressed alone is unable to functionally couple to adenylate cyclase or bind GTPγS in response to GABA, but substitution of the GABA_B2 N terminus with that of GABA_B1 abolishes GABA_B receptor function.a, Membranes prepared from cells expressing either GABA_B2 (GB2) alone (squares) or both GABA_B1 (GB1) and GABA_B2together (circles, triangles) were incubated with either GABA (circles,squares) or baclofen (triangles) and subsequently with [³⁵S]GTPγS. Data are presented as the percentage of increase in [³⁵S]GTPγS binding over basal in response to agonist and are expressed as means ± SEM (n = 3). b, Cells expressing either GABA_B2 alone or GABA_B1 and GABA_B2 together (symbols and legend as above) were incubated with forskolin and then either GABA or baclofen. Data are presented as the percentage of forskolin-stimulated cAMP levels remaining after incubation with agonist and are expressed as means ± SEM (n = 3). c, HEK293 cells were transiently transfected with GABA_B1N/2(GB1_N/2) and wtGABA_B1(GB1), and cell surface expression of GABA_Bsubunits was examined by immunofluorescence using either an anti-myc antibody for GABA_B1N/2 in the absence (i) or presence (ii) of detergent or an anti-GABA_B1 antibody for wtGABA_B1(iii). d, Representative FLIPR analysis showing no functional response to GABA in cells transiently transfected with G_qi5, GABA_B1N/2(GB1_N/2), and wtGABA_B1(GB1).

Fig. 8.

Proposed sideways signaling mechanism for the GABA_B receptor heterodimer. GABA_B1(orange) and GABA_B2(green) subunits form heterodimers via C-terminal coiled-coil interactions, as well as N-terminal and possibly transmembrane domain interactions. The agonist GABA binds to the ligand binding domain within the GABA_B1 N terminus, resulting in a conformational change in the GABA_B2 subunit. This activated state of the receptor heterodimer allows recruitment of G-proteins, at least in part via an interaction with the positively charged residues in il2 of GABA_B2 and subsequent activation of downstream signal transduction cascades.