Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning.
The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.
Rondard
Philippe
P
CNRS, UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France.
Huang
Siluo
S
Monnier
Carine
C
Tu
Haijun
H
Blanchard
Bertrand
B
Oueslati
Nadia
N
Malhaire
Fanny
F
Li
Ying
Y
Trinquet
Eric
E
Labesse
Gilles
G
Pin
Jean-Philippe
JP
Liu
Jianfeng
J
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D019295:Computational Biology; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018080:Receptors, GABA-B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D011134:Polysaccharides; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid