Locking the dimeric GABA(B) G-protein-coupled receptor in its active state.
G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.
Kniazeff
Julie
J
Laboratory for Functional Genomic, Department of Molecular Pharmacology, Centre National de la Recherche Scientifique Unité Propre de Recherche-2580, Montpellier Cedex 5, France.
Saintot
Pierre-Philippe
PP
Goudet
Cyril
C
Liu
Jianfeng
J
Charnet
Annie
A
Guillon
Gilles
G
Pin
Jean-Philippe
JP
D002460:Cell Line; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D018080:Receptors, GABA-B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D018755:GABA Agonists; D018756:GABA Antagonists; D018080:Receptors, GABA-B; D003545:Cysteine