Mapping the agonist-binding site of GABA(B) type 1 subunit sheds light on the activation process of GABA(B) receptors

The gamma -amino-n-butyric acid type B (GABA(B)) receptor is composed of tw o subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix rec eptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding d omain is likely to fold like bacterial periplasmic binding proteins that ar e constituted of two lobes that close upon ligand binding. Here, using mole cular modeling and site-directed mutagenesis, we have identified residues i n 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 car boxylic and amino groups of GABA, respectively, demonstrating the pivotal r ole 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 close d form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal rol e 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 bin ding domain of family 3 receptors is required for their activation.