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.

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.

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.

[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-HT1C, 5-HT1D and 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-HT-1D-like binding sites.