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We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [ Metabotropic glutamate receptors are G protein-coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. At present, eight mGluR 1 subtypes (mGluR1 through mGluR8) have been cloned and functionally expressed (1, 2). Based on their amino acid sequence homologies, pharmacology, and functional profiles, these subtypes are classified further into three groups. Members of group I (mGluR1 and -5) stimulate the activity of phospholipase C and mobilize intracellular Ca 2ϩ . Members of group II (mGluR2 and -3) and group III (mGluR4, -6, -7, -8) inhibit adenylyl cyclase. Despite the differences in primary structures and functional roles, all mGluRs feature a large conserved N-terminal extracellular domain, which is involved in the recognition of agonists and competitive antagonists (3-8).Most ligands for mGluRs were derived from amino acids and act at the conserved glutamate binding site (9). Recently, novel subtype-selective group I mGluR antagonists emerged that are structurally unrelated to amino acids and to each other. The first non-amino acid-like antagonist described was CPCCOEt (Fig. 1), a selective mGluR1 antagonist (10, 11). CPCCOEt inhibits receptor activity by a non-competitive mechanism which does not affect the binding affinity of glutamate (12, 13). Molecular characterization of the site of inhibition in mGluR1 revealed that CPCCOEt interacts with two non-conserved residues at the top of transmembrane (TM) helix VII (13). The first described selective mGluR5 antagonists, SIB-1757 and SIB-1893 (Fig. 1), are also unrelated to amino acids and were shown to act via a non-competitive mechanism (14).To address the question whether these structurally unrelated mGluR1 and mGluR5 antagonists interact with different sites of the mGluR subtypes or share a common binding site in the 7TM domain, we have studied the binding site and mode of action of the selective mGluR5 antagonist MPEP (15). MPEP is a novel derivative of SIB-1893 with nanomolar potency (Fig. 1); it is an effective antihyperalgesic in animal models of chronic inflammatory pain (16), a neuroprotectant in excitotoxin-induced striatal lesions (17) and an anticonvulsant in several epilepsy models (18). We generated a number of chimeric rec...

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre-and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTP␥S binding (EC50-values, 64 -290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (<10 M) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the bloodbrain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.G protein ͉ mGluR G protein-coupled receptors in vertebrates constitute a superfamily of 1,000-2,000 transmembrane heptahelical proteins that can be activated by a large number of extracellular signals such as photons, hormones, neurotransmitters, and growth and development factors. These receptors transduce and amplify cellular signals by the activation of G proteins, which in turn modulates cytoplasmic second-messenger and ion levels (1). Sequence comparison among the different G protein-coupled receptors revealed the existence of at least six receptor families (2). One of them, family 3, comprises receptors for extracellular calcium, pheromones, GABA, and L-glutamate. This receptor family is characterized by a large N-terminal extracellular domain that has been demonstrated by site-directed mutagenesis and x-ray crystallography to contain the binding site for orthosteric agonists (3-5). Metabotropic glutamate receptors (mGluRs) are members of family 3 and are activated by the major excitatory neurotransmitter of the mammalian brain, L-glutamate. The mGluRs act as important pre-and postsynaptic regulators of neurotransmission in the CNS, and there are at least eight subtypes (mGluR1 to m...

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56β, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.

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