Mutagenesis and modeling of the GABA(B) receptor extracellular domain support a Venus flytrap mechanism for ligand binding

The gamma-aminobutyric acid type B (GABA(B)) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled rec eptors (family 3), Sequence comparison revealed that, like metabotropic glu tamate receptors, the extracellular domain of the two GABA(B) receptor spli ce variants possesses an identical region homologous to the bacterial perip lasmic leucine-binding protein (LBP), but lacks the cysteine-rich region co mmon to all other family 3 receptors. A three dimensional model of the LBP- like domain of the GABA(B) receptor was constructed based on the known stru cture of LBP, This model predicts that four of the five cysteine residues f ound in this GABA(B) receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residue s and a decrease in the thermostability of the binding site after dithiothr eitol treatment. Additionally, Ser-246 was found to be critical for CGP6421 3 binding. Interestingly, this residue aligns with Ser-79 of LBP, which for ms a hydrogen bond with the ligand, The mutation of Ser-269 was found to di fferently affect the affinity of various ligands, indicating that this resi due is involved in the selectivity of recognition of GABA(B) re ceptor liga nds, Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for anta gonists. Such an effect of point mutations can be explained by the Venus fl ytrap model for receptor activation. This model proposes that the initial s tep in the activation of the receptor by agonist results from the closure o f the two lobes of the binding domain.