Type A y-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA Pl, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

GLP-1-(7-36 GLP-1-(7-36)-amide (GLP-1)1 is an incretin hormone that is secreted from the gastrointestinal tract in response to food intake and increases insulin secretion from pancreatic beta cells (1). The physiological action of GLP-1 gained considerable interest following the demonstration that GLP-1 acts on the pancreatic islet beta cells as a potent glucose-dependent insulin secretagogue (2-4). Despite previous structure-function studies of GLP-1 (5-8), no native analog of GLP-1 has been shown to possess potent antagonist activity.The GLP-1 receptor is a putative seven-transmembrane domain receptor (9) and belongs to the family of G-protein-coupled receptors that includes glucagon-secretin-vasoactive intestinal peptide receptors. GLP-1 binding to the pancreatic beta cell receptor induces an increase in intracellular cAMP levels (10).Exendin-4-(1-39) (exendin) was originally isolated from Gila monster venom (11) and is a member of the glucagon-secretinvasoactive intestinal peptide family of peptides. It has 48% amino acid sequence homology to glucagon and 50% homology to human GLP-1. An antagonist, exendin-4-(9 -39) (Ex9), was created by the deletion of 8 N-terminal amino acids from exendin (12). Subsequent work by Thorens et al. (13) showed that exendin acts directly on the GLP-1 receptor as an agonist, whereas Ex9 acts as an antagonist of the GLP-1 receptor and provided the first high potency antagonist of GLP-1. The purpose of this study is 2-fold: first, to analyze the transition from agonist to antagonist between exendin and Ex9, and second, to characterize the peptide domains of exendin that confer binding and thus antagonist activity by constructing exendin-(3-39)/GLP-1-(9 -36)-amide chimeras and by testing them for the retention of antagonist activity. MATERIALS AND METHODSPeptide Synthesis-Peptides were synthesized on a PAL resin solidphase support using activated Fmoc (N-(9-fluorenyl)methoxycarbonyl)-amino acids on a Milligen 9050 automated peptide synthesizer. Cleavage and deprotection of peptides were performed using 90% trifluoroacetic acid, 5% thioanisole, 3% anisole, and 2% ethanedithiol. Crude synthetic peptide mixtures were individually purified by preparative high pressure liquid chromatography. Purified peptides were quantitated by amino acid analysis.Plasmid Constructs-Full-length GLP-1 receptor cDNA isolated from rat pancreas (gift from Dr. Bernard Thorens, University of Lausanne, Lausanne, Switzerland) was subcloned in pSVbeta (CLONTEC, Palo Alto, CA) downstream of the SV40 promoter after replacing the ␤-galactosidase gene with a full-length GLP-1 receptor cDNA to obtain pSVGLPR.Cell Culture and Transfection-CHO cells that overexpress the human insulin receptor (CHO/HIRc cells) were trypsinized and resuspended in Ham's F-12 medium. Cells (10 6 cells in 800 l) were cotransfected with 10 g of HindIII-linearized pSVGLPR plasmid and 1 g of BamHI-linearized pSVHPH plasmid (conferring hygromycin resistance; American Type Culture Collection, Rockville, MD) by electroporation. Electroporation ...

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg Ϫ 1 ·min Ϫ 1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04 Ϯ 0.92 mmol/liter, P Ͻ 0.01) than saline-treated animals (

Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor-ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile. discriminate analysis ͉ hydrogen/deuterium exchange ͉ mass spectrometry T he estrogen receptors (ER␣ and ER␤) are important transcriptional regulators that mediate a number of fundamental processes including regulation of the reproductive system and the maintenance of skeletal and cardiovascular tone. As such, these receptors are the molecular targets of drugs used to treat diseases such as breast cancer and osteoporosis. Both beneficial and detrimental effects of ER ligands have been demonstrated in target tissues, thus tissue-selective ER ligands have been developed and are termed selective estrogen receptor modulators (SERMs). Traditional drug discovery programs for ER modulators most often involve the use of a receptor-binding assay as a primary screen to identify high-affinity ligands, followed by the use of in vitro cellbased assays to determine the functional activity of a given ligand (1). Compounds with the desired intrinsic properties for affinity and selective functional response are then evaluated for in vivo efficacy in animal models of the targeted disease. Although this drugdiscovery paradigm has been used successfully to identify most of the clinically-relevant SERMs discovered to date, the ability of in vitro biochemical and cell-based functional assays to translate to in vivo tissue selectivity has been limited. Cofactor recruitment assays have proven to be a useful tool to detect ligand-induced conformational changes for many nuclear receptors but can be less effective for profiling SERMs because the key coactivator interaction surface (AF-2) has been blocked by the ligand-induced repositioning of helix 12.Classical approaches for structural analysis of receptor-ligand interaction involve the use of x-ray crystallography or NMR spectroscopy. The importance of studying changes to protein dynamics during ER modulation has been demonstrated by Tamrazi et al. (2). In a serie...