Mapping the Agonist-binding Site of GABAB Type 1 Subunit Sheds Light on the Activation Process of GABABReceptors

Abstract

The gamma-amino-n-butyric acid type B (GABA(B)) receptor is composed of two subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix receptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding proteins that are constituted of two lobes that close upon ligand binding. Here, using molecular modeling and site-directed mutagenesis, we have identified residues in the GABA(B)1 subunit that are critical for agonist binding and activation of the heteromeric receptor. Our data suggest that two residues (Ser(246) and Asp(471)) located within lobe I form H bonds and a salt bridge with carboxylic and amino groups of GABA, respectively, demonstrating the pivotal role of lobe I in agonist binding. Interestingly, our data also suggest that a residue within lobe II (Tyr(366)) interacts with the agonists in a closed form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal role played by the GABA(B)1 subunit in the activation of the heteromeric GABA(B) receptor and are consistent with the idea that a closed state of the binding domain of family 3 receptors is required for their activation.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

“…Ligand Binding-The N-terminal domain is similar to bacterial amino acid-binding proteins and the N-terminal domain of mGluR and GABA B receptor subunits, which contain the neurotransmitter binding site (34,35). Still, previous studies on chimeric receptors (5) and soluble extracellular domains of AMPA receptor subunits (6,7,36) clearly exclude a direct participation of the N-terminal domain in agonist binding.…”

Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit

“…Modeling and site-directed mutagenesis studies indicate that ligands bind in the cleft that separates both lobes of GB1 VFT, as observed for ligand binding in many similar protein modules (23), including mGlu1 VFT (24,25). Antagonists are expected to prevent the closure of GB1 VFT (19), as observed in mGlu receptors (25,26). Conversely, agonists interact with residues from both lobes of GB1 VFT, and they stabilize a closed form of this domain (16,19).…”

“…The ligand-binding site of GB1 has been extensively studied (17)(18)(19)(20)(21)(22). Modeling and site-directed mutagenesis studies indicate that ligands bind in the cleft that separates both lobes of GB1 VFT, as observed for ligand binding in many similar protein modules (23), including mGlu1 VFT (24,25).…”

The activation mechanism of class-C G-protein coupled receptors

“…As mentioned above, the GABA B receptor is a heterodimer composed of both GABA B1 and GABA B2 subunits (Marshall et al, 1999). Whereas GABA B agonists and competitive antagonists interact in the GABA B1 VFTM (Galvez et al, 1999;Galvez et al, 2000), this subunit expressed alone does not give rise to consistent responses. Although this subunit is retained in the ER due to a retention signal in its C-terminal tail (Couve et al, 1998;Pagano et al, 2001), no function can also be observed with a subunit mutated in the ER retention signal , whereas robust responses can be obtained in the presence of GABA B2 .…”