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.

💡 Research Summary

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The paper addresses a long‑standing question in the field of class C G‑protein‑coupled receptors (GPCRs): how does agonist binding to the extracellular Venus‑flytrap (VFT) domain of the GABA(B)1 subunit trigger activation of the GABA(B)2 hepta‑helical (7TM) domain that couples to G proteins? To answer this, the authors devised a “glycan wedge scanning” strategy, in which they introduced consensus N‑glycosylation sequons (Asn‑X‑Ser/Thr) at selected positions of the VFT domains. Because an N‑linked glycan occupies roughly 1 nm of space, its presence can sterically block protein‑protein contacts without altering the primary sequence dramatically.

Computational identification of the dimer interface
Using the available crystal structures of the isolated VFTs, multiple sequence alignments of GABA(B)1 and GABA(B)2 orthologs, and surface‑accessibility calculations, the authors mapped a conserved interface that buries a relatively flat surface formed by β‑strands and loops from each subunit. Twelve residues on each side were deemed critical for dimerization; each was mutated to introduce an N‑glycosylation site.

Experimental validation – “interface wedges”
When the glycan was placed at any of these interface positions, co‑immunoprecipitation and surface‑biotinylation assays showed that the two subunits failed to associate and were retained intracellularly. Radioligand binding with