Milt Teitler - US grants

Altered sensitivity of multiple brain serotonin (5HT) receptors has been hypothesized in the etiology and/or treatment of anxiety, depression, sleep disorders, sexual behaviors, and psychosis. The roles that the multiple 5HT receptor systems play in brain function are still largely unknown, as are the neuronal mechanisms through which 5HT receptors alter cellular function. To better understand the nature of the brain's 5HT receptors have been developed and will be used in conjunction with in vivo manipulations to further our understanding of the role these receptors systems play in brain function and the effects of drugs. a) Preliminary evidence for the existence of a novel brain 5HT receptor, the 5HT1D receptors, has been uncovered. Detailed radioligand binding and adenylate cyclase studies of the 5HT1D receptor will be performed in various mammalian tissues including rat and human brain. b) in order to determine the susceptibility of the 5-HT1D receptors system to sensitivity changes, 5HT transmission in rats will be altered by acute and chronic administration of serotonin neurotoxins, receptor antagonists, agonists, anxiolytics, antidepressants, and electroconvulsive shock. Alterations in the amount of 5-HT1D receptors, the interaction of the 5HT1D receptor with its associated GTP-binding protein, and the coupling between 5HT1D receptors and brain adenylate cyclase activity due to these perturbations will be monitored. c) Preliminary studies have demonstrated that 125I-DOI labels either alternatively, a unique 5HT2 receptor with high affinity for 5HT2 agonists or, alternatively, a unique 5HT2 receptor distinct from that labelled by 3H-ketanserin. In order to address this question the distribution and regulation of the 125I-DOI labelled receptors will be determined and compared with the distribution and regulation of 3H-ketanserin (an antagonist) labelled 5-HT2 receptors in multiple brain regions. d) In order to investigate the role of the brain 5HT1A receptor systems in the actions of antidepressants and novel anxiolytics the affects of these drugs on 3H-BMY 7378 (the first 5HT1A receptor antagonist radioligand), and on the associated 5HT1A receptor-mediated inhibition of adenylate cyclase activity will be monitored.

biomedical equipment purchase;

The mechanism-of-action of clozapine, the prototypical atypical anti-psychotic drug, has been the subject of intense research aimed at revealing the key property of this drug that allows it to have superior antipsychotic activity relative to the more classical antipsychotic drugs. Evidence indicates that clozapine's high affinity interactions with the 5HT-2A receptor may be a key factor. In addition, clozapine has high affinity for the 5HT-2C, 5HT-6, and 5HT-7 receptors. Until recently it has been assumed that clozapine is a classical competitive antagonist at the 5HT-2A receptor. However, preliminary data in this proposal reveal that clozapine is an inverse agonist at the constitutively activated form of the 5HT-2A receptor, created by site-specific mutagenesis. In order to explore the possibility that clozapine's atypical activity may be related to its inverse agonist activity at the 5HT-2A receptor, the effects of a group of typical and atypical antipsychotic drugs on the constitutively activated 5HT-2A receptor will be determined. It is anticipated that if 5HT-2A inverse agonist activity is a critical factor in atypical drug properties, a correlation will be found between inverse agonist activity and atypical antipsychotic activity. In order to ascertain the specificity of the inverse agonist activity of clozapine at the 5HT-2A receptor, the effects of clozapine and other atypical antipsychotic drugs at constitutively activated forms of the 5HT-2C, 5HT-6 and 5HT-7 receptors will be determined. It is anticipated that these studies should confirm or refute the hypothesis that 5HT-2A receptor inverse agonist activity is critical to atypical antipsychotic activity, and may reveal a possible involvement of the 5HT-2C, 5HT-6 or 5HT-7 in atypical drug actions. It is also anticipated that similarities and differences in the pharmacology of the activated state of the four 5HT receptors studied will be revealed: this information may be important in future drug development aimed at developing inverse agonists for therapeutic purposes.

[unreadable] DESCRIPTION (provided by applicant): The 5HT2A receptor, a member of the G-protein-coupled receptor family (GPCR), appears to play a central role in controlling cognition and mood. 5HT2A agonists produce psychotomimetic effects and 5HT2A antagonism appears to contribute to the increased efficacy of atypical antipsychotic drugs. Therefore dysregulation of 5HT2A receptor activity may be involved in schizophrenia and/or depression. 5HT2A receptors are expressed in the synapses of brain regions critical for control of cognition and mood, i.e. layer V of the cortex and throughout the limbic system. This proposal involves the investigation of the structure and function of 5HT2A receptor expressing synapses on the molecular and cellular level. In order to discover novel brain proteins that interact with the 5HT2A receptor, the c-terminal tail of the 5HT2A receptor was used as the "bait" in a yeast two-hybrid assay system. Three "positive" clones, coding for regions of human brain proteins, were found to strongly interact with the 5HT2A receptor c-terminal tail. Based on homology searches of GENBANK, the positive clones code for partial sequences of "PDZ domain" proteins. This proposal involves the use of the yeast two-hybrid assay system to discover and investigate the properties of novel proteins that interact with the 5HT2A receptor and may participate in the structure and function of 5HT2A receptor-containing synapses. The specific aims are: l) to use the yeast two-hybrid assay we have developed to identify proteins in the adult human brain that can interact with the c-terminal tail of the human 5HT2A receptor; 2) to produce full-length clones from the partial clones identified from our yeast two-hybrid cDNA library; 3) to use the GST-fusion protein assay to validate the interaction of candidate proteins from the yeast two-hybrid assay outside of the yeast cell environment; 4) to use co-immunoprecipitation studies to demonstrate the interaction of the full-length 5HT2A receptor and candidate proteins in recombinant cell lines. These studies are designed to provide the foundation for an R01 application involving investigations of the structural and functional aspects of 5HT2A receptor synaptic molecular biology and may lead to the discovery of completely novel GPCR signal transduction mechanisms, possibly including receptor regulation mechanisms that may be disrupted in psychiatric disorders. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The 5HT1E serotonin receptor is one of fourteen serotonin receptors expressed in humans. It is especially highly expressed in human cortical brain tissue, indicating modulation of this receptor by drugs may produce robust effects on cognition and/or mood. There has been surprisingly little information on the inherent function of this receptor, presumably due to the lack of selective drugs to stimulate or antagonize the 5HT1E receptor. In order to advance the field of 5HT1E receptor pharmacology we propose to develop a high throughput screen (NTS) for selective agonists and antagonists for the human 5HT1E receptor. The specific aims of this proposal are to demonstrate that the recombinant cell line possesses the properties that will allow the application of HTS using SPA (Scintillation Proximity Assays) to the development of novel, specific agonists and antagonists for this understudied human brain serotonin receptor. Selective agonists and antagonists for the 5HT1E receptor should, at the least, provide information on the function of this highly expressed receptor, and may lead to novel therapeutics for brain dysfunction. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Novel mechanisms of modulating GPCR function may result in improved therapeutics for treating many diseases including schizophrenia and depression. Studies performed during the previous funding cycle of this grant revealed two unexpected effects of drugs on h5-HT6 and h5-HT7 receptor activity. 1. Two groups of inverse agonists have been discovered: "high affinity/high potency" inverse agonists with potencies that are predicted from their affinities for the receptor, and "high affinity/low potency" inverse agonists with potencies that are far lower than predicted from their binding affinities for the receptor. 2.The second unexpected result is that risperidone (a widely prescribed antipsychotic drug), 9-OH-risperidone (the active metabolite of risperidone), and methiothepin produce a rapid and potent inactivation of the native h5-HT7 receptor (inactivating antagonists). Therefore, this proposal involves two specific aims: 1) determine the mechanism of action of high potency vs. low potency inverse agonists at CAM h5-HT6 and 5-HT7 receptors;and 2) determine the mechanism(s) of action that results in risperidone's rapid and potent inactivation of the native h5-HT7 receptor. Specific aim"1 will be approached in three ways: a) detailed pharmacological analysis of the actions of inverse agonists at the CAM h5-HT6 and h5-HT7 receptors to determine if an allosteric mechanism may be involved;b) monitor the effects of inverse agonists on CAM h5-HT6 and h5- HT7 receptor internalization and beta-arrestin translocation;c) monitor the effects of inverse agonists on CAM h5-HT6 and h5-HT7 receptor associated MARK activity. Specific aim 2 will be approached in three ways: a) determine if inactivating antagonists interact irreversibly with the native h-5HT7 receptor in intact cell preparations;b) monitor the effects of inactivating antagonists on native h5-HT7 receptor internalization and beta-arrestin translocation, and c) monitor the effects of inactivating antagonist treatment on MARK activity in cells expressing native h5-HT7 receptors. The results from these studies may reveal novel mechanisms for modulating the functional state of h5-HT6 and h5-HT7 receptors, which may be applicable to many other GPCR. Dysfunctions of these modulating mechanisms may underly the psychopathology of various mental diseases. These studies may lead to the development of novel therapeutics for brain dysfunctions, including schizophrenia and depression.