Ligand-induced signal transduction within heterodimeric GABA(B) receptor

Abstract

gamma-aminobutyric acid type B (GABA(B)) receptors, G protein-coupled receptors (GPCRs) for GABA, are obligate heterodimers of two homologous subunits, GB1 and GB2. Typical for family C GPCRs, the N termini of both GB1 and GB2 contain a domain with homology to bacterial periplasmic amino acid-binding proteins (PBPs), but only the GB1 PBP-like domain binds GABA. We found that both GB1 and GB2 extracellular N termini are required for normal coupling of GABA(B) receptors to their physiological effectors, G(i) and G protein-activated K(+) channels (GIRKs). Receptors with two GB2 N termini did not respond to GABA, whereas receptors with two GB1 N termini showed increased basal activity and responded to GABA with inhibition, rather than activation, of GIRK channels. This GABA-induced GIRK current inhibition depended on GABA binding to the chimeric GB(1/2) subunit (the GB1 N-terminal domain attached to the heptahelical domain of GB2), rather than the wild-type GB1 subunit. Interestingly, receptors with reciprocal exchange of N-terminal domains between the subunits were functionally indistinguishable from wild-type receptors. We also found that peptide linkers between GB1 and GB2 PBP-like domains and respective heptahelical domains could be altered without affecting receptor function. This finding suggests that other contacts between the PBP-like and heptahelical domains underlie ligand-induced signal transduction, a finding likely to be relevant for all family C GPCRs.

Figure 1

Models of family C receptor signaling. (A) Model of mGluR1 activation based on the crystal structure of the mGluR1 PBP-like domain and illustrated here as in ref. . Glutamate binding stabilizes the closed conformation of the two PBP-like protomers, which results in shortening of the distance between the protomer C termini. It was hypothesized that as a result the two heptahelical domains are drawn closer, thus triggering G protein signaling (16); we refer to this scenario as the peptide-linker model of receptor activation because it relies on the movements of the peptide linker between the PBP-like and heptahelical domains. In mGluRs and CaR, but not GABA_B receptors, this linker contains a cysteine-rich (CR) domain. The receptor segments shown in white [the peptide linker, transmembrane segments (TM), and the intracellular C terminus (IC)] are represented schematically. The white spheres represent glutamate. (B) Two models of GABA_B receptor activation. The peptide-linker model of receptor activation (a) is based on the mGluR1 model. Note the presence of the C-terminal coiled–coil interaction in assembled GABA_B receptors. The direct contact model of receptor activation is shown in b. In this scenario, the ligand-binding associated conformational change of the PBP-like domain is transmitted to the rest of the receptor through direct contacts of the PBP-like and heptahelical domains. The GB1 subunit is shown in black and the GB2 subunit in gray; the light gray spheres represent GABA.

Figure 2

Chimeras GB_1/2 and GB_2/1 were functionally assayed by coexpression with GIRK1/GIRK2 channels in Xenopus oocytes. In this assay, binding of GABA to functional GABA_B receptors results in activation of inwardly rectifying K⁺ current; see Materials and Methods for experimental details. The I–V plots from representative cells (A–C) and summary plot for all of the cells tested (D) are shown. In this and subsequent figures, GB1 subunit is schematically represented in black and GB2 subunit in gray; the notch in GB1 subunit represents the GABA-binding site. In all figures, summarized data (which were not normally distributed) are represented as statistical box charts (the horizontal lines in the box denote 25th, 50th, and 75th percentile values, the error bars denote 5th and 95th percentile values, the asterisks denote 1st and 99th percentile values, and the square symbol in the box denotes the mean). For summary plot and statistical analysis, currents were measured at −120 mV; GABA responses are expressed as a percentage of basal GIRK currents recorded in 40K solution just before GABA application. (A) wt GABA_B receptors, composed of GB1 and GB2, respond to GABA application by GIRK current activation. (B) In cells coexpressing chimeras GB_1/2 and GB_2/1, GABA application results in GIRK activation that is no different from the activation mediated by wt receptors. (C) In contrast, chimera GB_1/2 coexpressed with GB1 inhibits GIRK current upon GABA application.

Figure 3

(A and B) Application of the competitive GABA_B receptor antagonist SCH 50911 to cells expressing wt GB1/GB2 receptors results in a slight inhibition of basal GIRK current, indicating that wt GABA_B receptors have low basal activity. (C and D) In contrast, in oocytes expressing GB1/GB_1/2 receptors, the magnitude of the SCH 50911-induced GIRK activation was 33.3% of the magnitude of the GABA-induced inhibition recorded in the same cells. Thus, GB1/GB_1/2 receptors have high basal activity, and GABA-mediated inhibition of GIRK current reflects the stabilization of the inactive receptor conformation. The I–V plots from representative cells are shown (A and C); data from cells tested with both drugs are summarized (B and D).

Figure 4

Mutating serine-246 to alanine disrupts the GABA-binding site in GB1, illustrated here by removal of the notch representing GABA-binding site. (A) The function of GB1/GB_1/2 receptors was not affected by the S246A mutation in the wt GB1 subunit. (B) In contrast, the analogous mutation in the GB_1/2 subunit completely abolished the inhibition of GIRK current mediated by GB1/GB_1/2 receptor complex. The I–V plots from representative cells (A and B) and summary plot (C) are shown. Data were statistically analyzed by Kruskal–Wallis one-way ANOVA on ranks, followed by the Dunn's posttest.

Figure 5

Amino acid sequences of ≈40-aa-long peptide linkers that connect the PBP-like domains with the transmembrane segments of GB1 and GB2 (A), the I–V plots from representative cells (B), and the summary plot (C) are shown. In the GB1_ad11 and GB2_ad11 mutant proteins, the 11-aa-long random coil peptide GGGASSASGG (boxed in gray) was inserted into GB1 and GB2 linkers at the positions shown, which resulted in linker extension. In the GB1_xg11 and GB2_xg11 mutant proteins, the GGGASSASGG peptide was inserted at the same position, but the preceding 11 residues of either GB1 or GB2 (underlined) were simultaneously deleted; the length of linkers was thus kept constant. Extension of GB1 and GB2 linkers had no effect on the GABA-mediated GIRK current activation, and 11 residues in the GB1 linker may be replaced with the random-coil peptide without functional consequences. Replacement of 11 residues in the GB2 linker prevented receptor cell surface expression (not shown).