No abstract

T.Galvez and B.Duthey contributed equally to this workRecent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The g-amino-n-butyric acid type B (GABA B ) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane a-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling ef®cacy. Conversely, although GB1 ECD is suf®cient to bind GABA B ligands, the ECD of GB2 increases the agonist af®nity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABA B receptor and highlight further the importance of the dimerization process in GPCR activation.

Ranvier nodes are flanked by paranodal regions, at the level of which oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region.

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.

The GABA(B) receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABA(B)-mediated effects, a single GABA(B) receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.