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

Class-C G-protein coupled receptors (GPCRs) represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca(2+), some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, class-C receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (ECD) responsible for agonist recognition and binding. In addition, it is now well accepted that these receptors are dimers, either homo or heterodimers. This complex architecture raises a number of important questions. Here we will discuss our view of how agonist binding within the large ECD triggers the necessary change of conformation, or stabilize a specific conformation, of the heptahelical domain leading to G-protein activation. How ligands acting within the heptahelical domain can change the properties of these complex macromolecules.

3-carboxy-4-phosphonocyclopentane amino acids: new metabotropic glutamate receptor ligands.

Two glutamic acid analogs (1 SR,3 RS,4 RS)- and (1 SR,3 SR,4 SR)-1-amino-4-phosphono cyclopentane-1,3-dicarboxylic acids (APCPD) have been synthesized. Pure E-(diethoxy-phosphoryl)-acrylic acid ethyl ester was obtained from ethyl propiolate, phenol and triethylphosphite. It was used as dienophile in a Diels-Alder reaction. Oxidation and cyclization afforded 3-(ethoxy-carbonyl)-4-(diethoxy-phosphoryl)-cyclopentanone. Bucherer-Bergs reaction and hydrolysis yielded APCPD-III and -IV which are inactive on mGlu1a receptor and antagonists on mGlu2 and mGlu8a receptors.

New probes of the agonist binding site of metabotropic glutamate receptors.

The (2S,4R)- and (2S,4S)-4-hydroxyglutamates activate cloned mGlu(1a), mGlu(2), and mGlu(8a) receptors with different potencies. Best results were obtained with the (2S,4S) isomer being almost as potent as glutamate on mGlu(1a)R and mGlu(8a)R. Data are interpreted on the basis of the binding site model and X-ray structure.

Pharmacological characterization of the rat metabotropic glutamate receptor type 8a revealed strong similarities and slight differences with the type 4a receptor.

In the brain, group-III metabotropic glutamate (mGlu) receptors mGlu(4), mGlu(7) and mGlu(8) receptors play a critical role in controlling the release process at many glutamatergic synapses. The pharmacological profile of mGlu(4) receptor has been studied extensively, allowing us to propose a pharmacophore model for this receptor subtype. Surprisingly, the activity of only a few compounds have been reported on mGlu(7) and mGlu(8) receptors. In order to identify new possibilities for the design of selective compounds able to discriminate between the members of the group-III mGlu receptors, we have undertaken a complete pharmacological characterization of mGlu(8) receptor and compared it with that of mGlu(4) receptor, using the same expression system, and the same read out. The activities of 32 different molecules revealed that these two mGlu receptors subtypes share a similar pharmacological profile. Only small differences were noticed in addition to that previously reported with S-carboxyglutamate (S-Gla) being a partial agonist at mGlu(4) receptor and a full antagonist at mGlu(8) receptor. These include: a slightly higher relative potency of the agonists 1S,3R and 1S,3S-aminocyclopentane-1,3-dicarboxylic acid (ACPD), S-4-carboxyphenylglycine (S-4CPG) and S-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG), and a slightly higher potency of the antagonists 2-aminobicyclo[3.1.0]hexane-2, 6-dicarboxylic acid (LY354740) and RS-alpha-methyl-4-phosphonophenylglycine (MPPG) on mGlu(8) receptor. When superimposed on the mGlu(4) receptor pharmacophore model, these molecules revealed three regions that may be different between the ligand binding sites of mGlu(8) and mGlu(4) receptors.

Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process.

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.

Extended glutamate activates metabotropic receptor types 1, 2 and 4: selective features at mGluR4 binding site.

To get an insight into the bioactive conformation of glutamic acid and its topological environment at the mGluR4 binding site, a pharmacophore model was constructed using molecular modeling. Agonists of known activities were used to run the Apex-3D program or to validate the resulting model. An extended glutamate conformer, two selective hydrophilic sites and bulk tolerance regions are disclosed. Selective features of mGluR1, mGluR2 and mGluR4 are discussed.

New perspectives for the development of selective metabotropic glutamate receptor ligands.

The metabotropic glutamate receptors are GTP-binding-protein (G-protein) coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. As such, these receptors are involved in a wide number of physiological and pathological processes. Within the last few years, new potent and selective agonists and antagonists as well as radioligands acting on these receptors have been developed. Molecular modeling studies revealed the structural features of the glutamate binding site, and will be useful for the design of more selective and potent ligands. More interestingly, recent data revealed new regulatory sites on the receptor protein, able either to decrease or potentiate the action of the endogenous ligand. No doubt that in the near future a multitude of new tools to modulate the activity of these receptors will be discovered, enabling the identification of the possible therapeutic applications for these new neuroactive molecules.