Christian Thomsen

The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins.

Receptors for the major excitatory neurotransmitter glutamate include metabotropic (G protein-coupled) and ionotropic (glutamate-gated ion channel) types. These receptors have large, presumably extracellular, amino-terminal domains. Sensitive sequence analysis techniques indicate that the metabotropic receptor extracellular domain is similar to bacterial periplasmic amino acid binding proteins. A structural model built using the observed similarity predicts a ligand-binding site, and mutants with conservative amino acid substitutions at this site are shown to have reduced ligand affinity. The metabotropic receptor extracellular domain is a member of a family of structural domains linked to a variety of receptor types, including ionotropic glutamate receptors.

Positive allosteric modulation of the human metabotropic glutamate receptor 4 (hmGluR4) by SIB‐1893 and MPEP

We have identified 2‐methyl‐6‐(2‐phenylethenyl)pyridine (SIB‐1893) and 2‐methyl‐6‐phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB‐1893 and MPEP enhanced the potency and efficacy of L‐2‐amino‐4‐phophonobutyrate (L‐AP4) in guanosine 5′‐O‐(3‐[35S]thiotriphosphate ([35S]GTPγS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB‐1893 and MPEP had no effects alone in GTPγS binding, effects were observed in the cell‐based cAMP assay due to media‐derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor‐specific effect since SIB‐1893 and MPEP had neither effects on mGluR2‐expressing cells nor on the parent BHK cell line. In [3H]L‐AP4 binding, a two‐fold decrease in KD but not in Bmax was observed with 100 μM SIB‐1893, whereas MPEP affected neither parameter. Finally, SIB‐1893 and MPEP failed to displace [3H]L‐AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

Effective Tumor Cell Death by σ-2 Receptor Ligand Siramesine Involves Lysosomal Leakage and Oxidative Stress

Acquired resistance to classic caspase-mediated apoptosis is a common problem for the treatment of human cancer. Here, we show that siramesine, a novel sigma-2 receptor ligand, effectively induces caspase-independent programmed cell death in immortalized and transformed cells of various origins. Siramesine-treated tumor cells displayed increased levels of reactive oxygen species, lysosomal membrane permeabilization, chromatin condensation, and shrinkage and detachment of cells. Lipid antioxidants (alpha-tocopherol and gamma-tocopherol), but not other tested antioxidants (butylated hydroxyanisol or N-acetyl cysteine), effectively inhibited siramesine-induced morphologic changes and cell death. Cathepsin B inhibitors (CA-074-Me and R-2525) conferred similar, but less pronounced protection, whereas ectopic expression of antiapoptotic protein Bcl-2, lack of wild-type p53 as well as pharmacologic inhibitors of caspases (zVAD-fmk, DEVD-CHO, and LEHD-CHO), calpains (PD150606), and serine proteases (N-tosyl-L-phenylalanine chloromethyl ketone and pefabloc) failed to protect cells against siramesine-induced death. Importantly, transformation of murine embryonic fibroblasts with activated c-src or v-Ha-ras oncogenes greatly sensitized them to siramesine-induced cytotoxicity. Furthermore, p.o. administration of well-tolerated doses of siramesine had a significant antitumorigenic effect in orthotopic breast cancer and s.c. fibrosarcoma models in mice. These results present siramesine as a promising new drug for the treatment of tumors resistant to traditional therapies.

Actions of phenylglycine analogs at subtypes of the metabotropic glutamate receptor family

The functional effects of phenylglycine analogs on metabotropic glutamate receptor (mGluR) subtypes mGluR1 alpha, mGluR2 and mGluR4 were examined. (S)-4-Carboxyphenylglycine (IC50 = 65 +/- 5 microM), (R,S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 155 +/- 38 microM) and (S)-3-carboxy-4-hydroxyphenylglycine (IC50 = 290 +/- 47 microM) competitively antagonized glutamate-stimulated phosphoinositide hydrolysis in baby hamster kidney (BHK) cells stably expressing mGluR1 alpha. (S)-4-Carboxyphenylglycine and (R,S)-alpha-methyl-4-carboxyphenylglycine competitively antagonized glutamate-induced inhibition of forskolin-stimulated cAMP-formation in BHK cells stably expressing mGluR2 with IC50 values of 577 +/- 74 microM and 340 +/- 59 microM, respectively. (R,S)-4-carboxy-3-hydroxyphenylglycine, (R)-3-hydroxyphenylglycine and (S)-3-carboxy-4-hydroxyphenylglycine were agonists at mGluR2 with EC50 values of 48 +/- 5 microM, 451 +/- 93 and 97 +/- 12 microM, respectively. In parallel experiments, no activities of these phenylglycine analogs at mGluR4 were observed. The present report demonstrates that phenylglycine analogs possess differential functional activities at subtypes of the mGluR family.

L-2-amino-4-phosphonobutyrate (L-AP4) is an agonist at the type iv metabotropic glutamate receptor which is negatively coupled to adenylate cyclase

Abstract Glutamate and L-AP4 inhibited forskolin-stimulated cyclic AMP (cAMP) production in baby hamster kidney (BHK) cells transfected with the type IV metabotropic receptor (mGluR4). In situ hybridization revealed a high level of mRNA for the mGluR4 in the entorhinal cortex, but not in the dentate gyrus. These data demonstrate that mGluR4 receptors arc negatively coupled to the cAMP cascade, and suggest that the mGluR4 receptor may be the previously described presynaptic L-AP4 receptor.

A Comparison of Two Alternatively Spliced Forms of a Metabotropic Glutamate Receptor Coupled to Phosphoinositide Turnover

Abstract: A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1α and 1β receptors (mGluR1α and mGluR1β) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1β receptor is an alternatively spliced form of mGluR1α with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor‐specific antipeptide antibodies showed that mGluRIa migrated with an Mr= 154, 000, whereas mGluR1β migrated with an Mr= 96, 000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1α receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1β was distributed diffusely throughout the cell. Agonist activation of the mGluR1α and the mGluR1β receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans‐(+)‐1‐aminocyclopentane‐1, 3‐dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1α showed pertussis toxin‐sensitive and insensitive components, whereas the mGluR1β response displayed only the toxin‐insensitive component. The mGluR1α and mGluR1β receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.

A pharmacological characterization of the mGluR1α subtype of the metabotropic glutamate receptor expressed in a cloned baby hamster kidney cell line

The pharmacological specificity of the mGluR1 alpha subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [3H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112 +/- 105% of basal), glutamate (1061 +/- 70% of basal), ibotenate (1097 +/- 115% of basal) and beta-N-methylamino-L-alanine (BMAA) (1010 +/- 104% of basal). In contrast, the maximal stimulation of PI-hydrolysis by (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) was only 673 +/- 78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25 +/- 4% inhibition) by L-2-amino-3-phosphonopropionic acid and partially blocked (44 +/- 7%) by pertussis toxin treatment. Saturation binding studies with [3H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1 alpha subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (Kd = 296 nM, Bmax = 0.8 pmol/mg protein). In competition experiments, [3H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.

Probing the Ligand-binding Domain of the mGluR4 Subtype of Metabotropic Glutamate Receptor

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool.  Although the conversion of Arg78, Ser159, or Thr182 to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist l-[3H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser157 and Ser160) or a relatively modest effect (Ser181) on ligand affinity compared with the Arg78, Ser159, and Thr182 mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog l-serineO-phosphate (l-SOP) was “docked” into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser159 and Thr182 of mGluR4 form hydrogen bonds with the α-carboxyl and α-amino groups on l-SOP, respectively, whereas Arg78 forms an electrostatic interaction with the acidic side chains of l-SOP or glutamate. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.

(S)‐Carboxy‐3‐Hydroxyphenylglycine, an Antagonist of Metabotropic Glutamate Receptor (mGluR)1a and an Agonist of mGluR2, Protects Against Audiogenic Seizures in DBA/2 Mice

Abstract: The in vivo anticonvulsant effects and in vitro metabo‐tropic glutamate receptor selectivity of (S)‐4‐carboxy‐3‐hydroxy‐phenylglycine [(S)‐4C3HPG] were examined. Intracerebroventricular injection of (S)‐4C3HPG dose‐dependently antagonized audiogenic‐induced clonic and tonic convulsions in DBA/2 mice with ED60 values of 76 and 110‐nmol per mouse, respectively. (S)‐4C3HPG dose‐dependently inhibited the spontaneously evoked epileptic spikes in a cingulate cortex‐corpus callosum slice preparation. (SJ‐4C3HPG displaced the binding of [3H]glutamate in membranes prepared from baby hamster kidney (BHK) cells expressing the metabotropic glutamate receptor mGluR1a with an EC50 of 5 β 1 uM. (S)‐4C3HPG dose‐dependently antagonized glutamate‐stimulated phosphoinositide hydrolysis in BHK cells expressing mGluR 1a with an IC50 of 15 β 3 μM. (S)‐4C3HPG was, however, an agonist at mGluR2 with an EC60 of 21 β 4 μM for inhibition of forskolin‐stimulated cyclic AMP formation in BHK cells expressing the mGluR2. (S)‐4C3HPG had no effects at mGluR4a. These data suggest that the anticonvulsant action of (S)‐4C3HPG is mediated by combined antagonism of mGluRIa and agonism of mGluR2. These results suggest the importance of mGluR1a and/or mGluR2 in the control of epileptic activity.

l‐Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l‐Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l‐glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l‐Serine‐O‐sulfate was an agonist at mGluR1a (EC50 = 70 µM), mGluR2 (EC50 = 25 µM), and mGluR4 (EC50 = 324 µM). l‐Cysteine sulfinate, 1‐aminocyclobutane‐trans‐1,3‐dicarboxylate, l‐cysteine, and dl‐threo‐3‐methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC50 values of 43, 64, 463, and 488 µM, respectively, and displaced l‐[3H]glutamate binding from membranes prepared from these cells with respective IC50 values of 48, 44, 79, and 139 µM. However, d‐aspartate,l‐trans‐pyrrolidine‐2,4‐dicarboxylate, l‐threo‐3‐hydroxyaspartate, and l‐aspartate‐β‐hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC50 values of 73, 54, 57, and 430 µM) but did not displace l‐[3H]glutamate binding. These compounds inhibited Na+‐dependent l‐glutamate uptake into baby hamster kidney cells with IC50 values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l‐glutamate transporter, was significantly attenuated in the presence of l‐glutamate decarboxylase (EC 4.1.1.15) or l‐alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl‐trans‐pyrrolidine‐2,4‐dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four‐ to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l‐glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.