Allosteric interactions between GABAB1 subunits control orthosteric binding sites occupancy within GABAB oligomers

Abstract

The GABA receptor was the first G protein-coupled receptor identified as an obligate heterodimer. It is composed of two subunits, GABA containing the agonist binding site and GABA responsible for G protein activation. The GABA receptor was found to associate into larger complexes through GABA-GABA interactions, both in transfected cells and in brain membranes. Here we assessed the possible allosteric interactions between GABA heterodimers by analyzing the effect of mutations located at the putative interface between the extracellular binding domains. These mutations decrease, but do not suppress, the Förster resonance energy transfer (FRET) signal measured between GABA subunits. Further analysis of one of these mutations revealed an increase in G protein-coupling efficacy and in the maximal antagonist binding by approximately two-fold. Hypothesizing that a tetramer is an elementary unit within oligomers, additional FRET data using fluorescent ligands and tagged subunits suggest that adjacent binding sites within the GABA oligomers are not simultaneously occupied. Our data show a strong negative effect between GABA binding sites within GABA oligomers. Accordingly, GABA receptor assembly appears to limit receptor signaling to G proteins, a property that may offer novel regulatory mechanism for this important neuronal receptor. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Rearrangement of the transmembrane domain interfaces associated with the activation of a GPCR hetero-oligomer

“…We were not surprised to observe GB1-GB1 cross-linking, when using a GB1 subunit carrying a Cys residue, as it was known that the GABA B receptor can associate into larger complexes likely through GB1-GB1 interaction 12,35 (Fig. 3a–b).…”

“…(ii) Allosteric interactions between the seven transmembrane helices (7TMs) of GB1 and GB2 lead to improved coupling efficacy of GB2 16 . (iii) GABA B receptors have the propensity to form stable hetero-oligomers organized through interactions between the GB1 subunits 12,28,33–35 (Fig. 1b).…”

“…(iv) Allosteric interactions between the heterodimeric units within such oligomers have been identified. These interactions allow a single heterodimer to bind ligand and activate G-proteins, within a tetrameric entity 12,35 . Despite this clear evidence of allosteric interactions between the subunits of the GABA B oligomer, and the known structure of the active and inactive heterodimeric extracellular domain 36 , little is known about 7TM structure.

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The organizing principle of GABAB receptor complexes: Physiological and pharmacological implications

“…Assembly of GB1 with GB2 releases PRAF2, which allows the heterodimeric receptor to exit the endoplasmic reticulum and to traffic to the cell surface. GB1/GB2 heterodimers at the plasma membrane can form transient higher‐order complexes via interaction of their GB1 subunits . Higher‐order complexes assemble by random collision of heterodimers in an activity‐independent manner.…”

“…Consequently, higher‐order complexes are more abundant at higher densities of heterodimers . Assembly into higher oligomers limits receptor signalling via G proteins because neighbouring G protein binding sites cannot be simultaneously occupied …”