This work was wppwted by grants from the CNRS, CEE (B102-CT93-0243), DRET (9 l/161) the Human Frontier5 Science Projiram (RG 5792B), and Bayer Company (Germany). This work has been d&e as part of the Bi oAveni n program supported by Rhone-Poul enc with the participation of the French Ministrv of Research and the French Ministrv of Industrv. J.G. was i supported by an IPSEN and then a Spnni\h governmen; fellowship\. We thank Dra. L. Journot and B. Chin! for critical reading of the manuscript, Dr. M. Lafon-Canal for the determlnatmn of the Glu ct&entrations, Dr. b. Jouhert for her hel p i n the confocal microscope cxperlmenta, and Dr. C. Romano for the gili of the mGluR5 antihwly Confocal microscopy experiments have been realired using the l'aulities of the Centre RCgional d'lmagerie Cel l ul ai re (C.R.I.C.). We thank also M. Passama and L. Charvet for their hel p i n the iconography. Correspondence shoul d be addressed to Dr. Jean-Phi l i ppe Pin, MCcanismes Mol&xdaires des Communications Cellulaires. 1JPR 902%CNRS, CCIPE, Rue de l a Cerdoni l l e 34OY4 Mtrntpellier Cetlex OS. France.

The ␥-aminobutyric acid type B (GABA B ) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABA B receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteinerich region common 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 structure of LBP. This model predicts that four of the five cysteine residues found in this GABA B receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABA B receptor ligands. 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 antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

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