Abstract
The gamma-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor.
Highlights
␥-Aminobutyric acid (GABA)3 is a major inhibitory neurotransmitter of the central nervous system that activates two types of receptor, the ionotropic GABAA/C receptors, to pro
Expression and Purification of the Extracellular Regions of the GABAB Receptor—Fig. 1 shows diagrams of the recombinant proteins of the GABAB receptor extracellular region (ECR) used in the baculovirus infection experiments
Determination of the C-terminal End of GBR1 Extracellular Region That Possesses Ligand Binding Activity—In this study, we determined the essential region of the GBR1 for ligand binding
Summary
Materials—Baclofen, CGP54626, and [3H]CGP54626 (53.31 Ci/mmol) were purchased from Tocris (UK). The PCR product was cloned using the BamHI and XbaI sites of pBlueScript, which contains a hemagglutinin signal sequence followed by a FLAG epitope (DYKDDDDK) tag just upstream of the BamHI site. For the longer GBR1-ECR2 and -ECR3 constructs, PCRs were performed with a forward primer with an MfeI site and reverse primers designed at distinct positions with a stop codon followed by a NheI site. Protein Expression and Purification—HighFive cells were cultured in a monolayer at 27 °C in Express Five serum-free medium (Invitrogen) supplemented with 18 mM L-glutamine. The supernatant containing the GBR2-ECR protein was directly applied to nickel-Sepharose (Amersham Biosciences) packed in a disposable column (Bio-Rad). Density Gradient Centrifugation—The GBR1-ECR3 protein, partially purified by the anti-FLAG M2 column, the GBR2-ECR protein, partially purified by the nickel column, and a mixture of both proteins were sedimented through 15–35% sucrose gradients formed with the high salt buffer. Proteins were visualized and quantified using an ECL detection kit (Amersham Biosciences)
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