We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a G i-coupled designer receptor in hippocampal neurons (hM 4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.cell engineering ͉ molecular evolution ͉ receptorome B ecause of the assorted cellular responses directed by them, their number, and the ease of which they are pharmacologically screened, the superfamily of G protein-coupled receptors (GPCRs) is one of the most therapeutically important targets in the proteome (1). However, the potential of this family is restricted by our ability to assess their function, which currently involves transgenic, knockout, and/or in vivo studies with selective drugs. Genetic studies are frequently limited to loss-of-function phenotypes, whereas nonselectiveness of a drug often interferes with interpretation of pharmacological studies. Knowledge of the roles of the individual family members is being bolstered by the ongoing creation of knockout mice for many GPCRs. Selective activation of individual GPCR subtypes in a defined tissue, in either a knockout or wild-type animal, is currently problematic but, if possible, would serve to complement present findings by providing novel insights into disease states resulting from overstimulation of certain signaling pathways.One approach to this problem has been to rationally modify receptors to favor synthetic over natural substrate/ligand recognition, and subsequently, these mutant proteins have been used as bio-tools to study protein function in complex biological environments (2, 3). At the forefront of such modified GPCRs is Ro1, a G i/o -coupled opioid receptor activated by a synthetic but not a native ligand, which has been conditionally expressed in transgenic mice to study cardiac function after its selective activation (4). Such mutant receptors, like Ro1, have been classified as receptors activated solely by synthetic ligands (RASSLs), because they are activated by synthetic ligands but not by their endogenous ligands (5). RASSLs, as in the case of Ro1, have been demonstrated to be valuable tools (4, 6); however, because the synthetic ligand frequently has high affinity and/or potency at the native receptor (5,7,8), this pote...

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-D-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca 2ϩ assays (EC 50 ϭ 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 M) and competed for binding of3 H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) ϭ 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED ϭ 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED ϭ 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED ϭ 1 mg/kg i.p.) and reduced impulsivity in the fivechoice serial reaction time test (MED ϭ 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.The metabotropic glutamate receptor (mGlu) receptor family includes eight G-protein-coupled receptor (GPCR) subtypes classified on the basis of structural homology, Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.136580.ABBREVIATIONS: mGlu, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; PAM, positive allosteric modulator; CPPHA, 2, NMDA, MPEP,

No abstract

Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein– protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1–TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1–TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.

Neurobiology. In the article ''ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae '' by Steve A. N. Goldstein, Laura A. Price, David N. Rosenthal, and Mark H. Pausch, which appeared in number 23, November 12, 1996, of Proc. Natl. Acad. Sci. USA (93, 13256-13261), the authors request that the following sequence correction be noted. We have found errors in the ORK1 nucleotide sequence reported in this work. The correct sequence extends the ORF and predicts a protein of 1001 residues; the correct nucleotide and predicted protein sequences are deposited under the GenBank accession no. U55321. The errors do not otherwise alter the conclusions of the paper. We are grateful to Noam Zilberberg (Yale Univ. School of Medicine, New Haven, CT) for his efforts to establish the correct sequence.Neurobiology. In the article "A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia" by Hiroaki Kawasaki, Gregory M. Springett, Shinichiro Toki, Juan J. Canales, Patricia Harlan, Justin P. Blumenstiel, Emy J. Chen, I. Amy Bany, Naoki Mochizuki, Amy Ashbacher, Michiyuki Matsuda, David E. Housman, and Ann M. Graybiel, which appeared in number 22, October 27, 1998, of Proc. Natl. Acad. Sci. USA (95, 13278-13283), due to a printer's error, the gene CalDAG-GEFII was referred to incorrectly in three places: in the heading of the second paragraph of Materials and Methods, in the first line of the Abbreviations footnote, and in line 11 of the second paragraph on page 13282. 318Corrections Proc. Natl. Acad. Sci. USA 96 (1999) Communicated by Vincent T. Marchesi, Yale University, New Haven, CT, August 21, 1996 (received for review July 5, 1996 ABSTRACT A K ؉ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K ؉ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K ؉