Abstract
The gamma-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.
Highlights
The ␥-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2)
Whereas metabotropic glutamate (mGlu) and Ca2ϩ-sensing receptors exist as homodimers, the GABAB receptor is a heterodimer composed of two homologous subunits, GABAB1 (GB1) and GABAB2 (GB2) (4 –7)
This is due partly to GB2 because its coexpression with GB1 results in a 10-fold increase in agonist affinity [4]. This effect of GB2 does not result from the targeting of GB1 to the cell surface and so to a mature glycosylation state because a GB1 mutant able to reach the cell surface alone (GB1ASA, in which the endoplasmic reticulum retention signal RSRR is mutated into ASAR) still displayed low agonist affinity at the cell surface (Fig. 1b and the above-described constructs alone or in combination (Table I))
Summary
The ␥-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Coexpression of GB1 with GB2/1, a chimeric subunit composed of GB2 VFT and GB1 HD, or the replacement of GB1 HD with GB2 HD in chimeric GB1/2 resulted in increased GABA affinity (Fig. 1b and Table I). ⌬GB1 and GB1GPI bound a competitive and membrane non-permeant radiolabeled antagonist ([125I]CGP64213), and this binding could be displaced by GABA, demonstrating that these constructs retained their ability to bind GABAB ligands (Fig. 2d and Table I).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
