M 1 muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M 1 receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M 1 receptor affinity for acetylcholine. We found that activation of the M 1 receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M 1 receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M 1 receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M 1 receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M 1 mAChR subtype may ameliorate impairments in cognitive function.

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Previous clinical and animal studies suggest that selective activators of M 1 and/or M 4 muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M 1 or M 4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M 4 mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d], that are potent and selective positive allosteric modulators of M 4 . VU0152099 and VU0152100 had no agonist activity but potentiated responses of M 4 to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M 4 reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M 4 plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M 4 may mimic some of the antipsychotic-like effects of less selective mAChR agonists.To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M 1 -M 5 ) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Wess, 1996). Of these, M 1 and M 4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Bymaster et al., 2002;Messer, 2002;Raedler et al., 2007). In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M 2 and M 3 mAChRs (Bymaster et al., 2003a,b).

Arrestin regulates almost all G protein-coupled receptor (GPCR)-mediated signaling and trafficking. We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gbetagamma complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of alpha2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.

G protein-coupled receptors (GPCRs) modulate diverse physiological and behavioral signaling pathways by virtue of changes in receptor activation and inactivation states. Functional changes in receptor properties include dynamic interactions with regulatory molecules and trafficking to various cellular compartments at various stages of the life cycle of a GPCR. This review focuses on trafficking of GPCRs to the cell surface, stabilization there, and agonist-regulated turnover. GPCR interactions with a variety of newly revealed partners also are reviewed with the intention of provoking further analysis of the relevance of these interactions in GPCR trafficking, signaling, or both. The disease consequences of mislocalization of GPCRs also are described.

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Activators of M 1 muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M 1 -active compounds has resulted in nonselective activation of the highly related M 2 to M 5 mAChR subtypes, which results in doselimiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M 1 with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentrationresponse curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,progressive shifts in ACh affinity at M 1 that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M 1 activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M 1 receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M 1 agonist, 1-[1Ј-(2-tolyl)-1,4Ј-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M 1 -mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M 1 is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M 1 potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.The psychotic and cognitive symptoms of neuropsychiatric disorders such as schizophrenia and Alzheimer's disease (AD) remain serious unmet medical challenges. Patients with schizophrenia exhibit a constellation of symptoms that include positive, negative, and cognitive symptom clusters. Although current antipsychotic agents are effective in reducing positive symptoms such as hallucinations and delusions in most patients, negative symptoms such as anhedonia and blunted affect, as well as deficits in cognitive function, are not effectively treated with current medications (Vohora, 2007). In addition to the unmet medical needs of schizophrenia, the devastating cognitive and neuropsychiatric symptoms characteristic of AD present urgent needs for new therapeutic interventions (Saddichha and Pandey, 2008).