Cathy D. Mahle

Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells

Galanin is a ubiquitous neuropeptide that regulates a wide array of physiological processes via interaction with specific G protein-coupled receptors. A rat galanin receptor cDNA was cloned from the Rin14B insulinoma cell line. The isolated cDNA encodes a 346 amino acid G protein-coupled receptor that is 92% identical to the recently reported human GALR1 galanin receptor. [125I]Galanin binds with high affinity to two receptor states in COS1 cell membranes containing the rat GALR1 receptor, consistent with coupling of the receptor to a G protein in these membranes. N-terminal galanin fragments and the putative galanin receptor antagonists galantide, C7, M35 and M40 bind with high affinity to the rat GALR1 receptor. In contrast, C-terminal galanin fragments do not bind to this receptor. Galanin inhibits basal and forskolin-stimulated cAMP formation in CHO cells expressing the rat GALR1 receptor via a pertussis toxin-sensitive G protein. The GALR1 receptor is expressed in rat spinal cord, small intestine, Rin14B insulinoma cells and several brain regions, particularly ventral hippocampus, amygdala, supraoptic nucleus, hypothalamus, thalamus, lateral parabrachial nucleus and locus coeruleus. Cloning of the rat GALR1 galanin receptor cDNA will permit many new experimental strategies to be applied to studies of the structure and function of galanin receptors.

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein‐coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel1a and Mel1b melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein‐coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N‐linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS‐1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Cloning and characterization of the rat 5-HT5B receptor : evidence that the 5-HT5B receptor couples to a G protein in mammalian cell membranes

A gene encoding a novel G protein‐coupled 5‐hydroxytryptamine (5‐HT) receptor, termed 5‐HT5B, was cloned. The ligand binding profile of this receptor is distinct from that of other cloned 5‐HT receptors. The 5‐HT5B receptor couples to a G protein in COS1 cell membranes; however, activation of the 5‐HT5B receptor does not appear to alter either cAMP accumulation or phosphoinositide turnover in a variety of fibroblast cell lines. In the rat brain, 5‐HT5B gene expression occurs predominantly in the medial habenulae and hippocampal CA1 cells of the adult. Little expression is seen during embryonic development.

A Novel Long-Acting Selective Neuropeptide Y2 Receptor Polyethylene Glycol-Conjugated Peptide Agonist Reduces Food Intake and Body Weight and Improves Glucose Metabolism in Rodents

Selective activation of the neuropeptide Y (NPY)2 receptor to suppress appetite provides a promising approach to obesity management. A selective NPY2 polyethylene glycol-conjugated (PEGylated) peptide agonist is described that consists of a peptide core corresponding to residues 13 to 36 of human peptide YY (PYY) and a nonpeptidic moiety (2-mercaptonicotinic acid) at the peptide N terminus that is derivatized with 20-kDa monomethoxypolyethylene glycol. The PEGylated peptide elicits a dose-dependent reduction in food intake in lean C57BL/6 mice and Wistar rats that persists for 72 and 48 h, respectively. The effect on food intake in lean C57BL/6 mice is blocked by the selective NPY2 antagonist BIIE0246 (N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide formate). A dose-dependent reduction in body weight in diet-induced obese (DIO) mice is seen following daily dosing for 14 days. The reduction in body weight is sustained following dosing for 40 days, and it is accompanied by an increase in plasma adiponectin. Improvements in glucose disposal and in plasma insulin and glucose levels that are risk factors for type II diabetes are observed following once-daily subcutaneous dosing in DIO mice. The results provide evidence from two animal species that the long-acting selective NPY2 peptide agonist has potential for obesity management.

Reduced bicuculline response and GABAA agonist binding in aged rat hippocampus

Extracellular field recordings from CA1 pyramidal cells in the rat hippocampal slice preparation were used to examine the effects of age on gamma-aminobutyric acid (GABA)-mediated recurrent inhibition. The actions of bicuculline (1-100 microM), a GABAA antagonist, were assessed in slices from young (1-3 months) and aged (26 months) Fischer 344 rats. Pre-drug population spike amplitudes were smaller in slices from aged rats. Bicuculline increased population spike amplitudes in slices from both age groups, but slices from young rats were more sensitive to the antagonist. Bicuculline also produced multiple population spikes in slices from both age groups, however the increase in population spike burst durations was much greater in slices from young rats than in slices from aged rats. Agonist radiolabeled GABAA binding site density was significantly decreased in hippocampal tissue from aged rats. Our results suggest there is a reduction in GABAergic inhibition in hippocampal slices from aged rats, possibly mediated by a decrease in GABAA receptors.

Bivalent indoles exhibiting serotonergic binding affinity

Abstract A series of bis-5-carboxamidoindoles were prepared and examined in a number of serotonin assays (5HT 1A , 5HT 1D , 5HT 1E , and 5HT UT). 1 They were found to exhibit good affinity for the 5HT 1A and 5HT 1D receptor subtypes and to inhibit the 5HT uptake sites. Optimal 5HT 1D potency was achieved for bivalent analogs 5f and 5g , whose linkers spanned 7 and 8 alkyl units. Analogs with longer alkyl chain linkers (n= 10, 12), 5h and 5i , exhibited no selectivity for the 5HT 1D receptor over the 5HT 1A receptor.

Characterization of human 5-HT1 receptors expressed in Sf9 insect cells

Four human 5-HT receptor subtypes (5-HT1A, 5-HT1D alpha, 5-HT1D beta and 5-HT1E) have been expressed in Sf9 insect cells. All four human 5-hydroxytryptamine receptors produced by Sf9 cells had the expected pharmacological properties. Surprisingly, levels of expression of these receptors were relatively low (1-5 pmol/mg protein). High affinity agonist binding to the four 5-hydroxytryptamine receptors was reduced to different extents by guanine nucleotides and/or NaCl. This suggests that the nature of receptor-G protein coupling and/or the predominant conformational state of the receptors in Sf9 cell membranes varies among the different receptors. Activation of all four receptors inhibited forskolin-stimulated cAMP formation in intact Sf9 cells. Expression of 5-hydroxytryptamine receptors in Sf9 cells should be useful for purification of these receptors, for studies of post-translational modification and for pharmaceutical screening.

[3H]-5-carboxamidotryptamine labels 5-HT1D binding sites in bovine substantia nigra.

1 [3H]‐5‐hydroxytryptamine (5‐HT) has been shown to radiolabel at least five types of 5‐HT binding sites in mammalian brain tissue, 5‐HT1A, 5‐HT1B, 5‐HT1C, 5‐HT1D and 5‐HT1E (Frazer et al., 1990). Selective masking of 5‐HT1A and 5‐HT1C receptors, has uncovered binding sites which display both high (5‐HT1D) and low (5‐HT1E) affinity for 5‐carboxamidotryptamine (5‐CT). By utilizing [3H]‐5‐CT we have eliminated a portion of the complex binding (5‐HT1E) seen when [3H]‐5‐HT is used as a radioligand. 2 [3H]‐5‐CT binding to 5‐HT1D sites in bovine substantia nigra was rapid, reversible and saturable, displaying high affinity (Kd = 0.38 ± 0.04 nm) and low non‐specific binding (> 90% specific binding). 3 In bovine substantia nigra, [3H]‐5‐CT labelled an equivalent number of binding sites to [3H]‐5‐CT (403 ± 18 and 362 ± 20 fmol mg−1 protein, respectively) and binding was sensitive to guanine nucleotides. 4 A linear correlation (r2 = 0.99) existed between the potency of compounds to displace [3H]‐5‐HT and [3H]‐5‐CT in bovine substantia nigra. 5 Therefore, [3H]‐5‐CT is a novel radioligand for the examination of 5‐HT1‐like binding sites, which under proper experimental conditions can be used to radiolabel selectively 5‐HT1D‐like binding sites.

Melatonin receptors: potential targets for central nervous system disorders.

The pineal hormone melatonin has become the subject of considerable speculation in both the scientific and lay press. Media coverage, coupled with scientific interest fuelled by the recent molecular cloning of a family of melatonin receptors, has led to a renaissance in melatonin research. While numerous physiological effects have been attributed to melatonin, the lack of selective agonists and antagonists for individual melatonin receptor subtypes has hampered progress towards the elucidation of the roles of these receptors. This review focuses on the molecular and pharmacological characterisation of melatonin receptors, the possible clinical utility of melatonin receptor ligands, and the progress towards the identification of selective ligands for these receptors.