Determination of the minimal functional ligand-binding domain of the GABA_B(1b) receptor

In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA_B receptors (where GABA stands for γ-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA_B receptors are assembled from two subunits GABA_B(1) (GABA_B receptor subtype 1) and GABA_B(2). For the GABA_B(1) subunit, which binds the neurotransmitter GABA, two variants GABA_B(1a) (GABA_B receptor subtype 1 variant a) and GABA_B(1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA_B(1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA_B(2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA_B(1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA_B(1) can be used for further functional or structural investigations.