Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase.

Serotonin modulates a wide range of physiological functions by activating multiple receptors, which are coupled to various effector systems. Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors, we have isolated from a mouse brain library a cDNA encoding a new 5-HT receptor, 5-HTx, that activates adenylate cyclase. Amino acid sequence comparisons revealed that the 5-HTx receptor was a distant relative of previously cloned 5-HT receptors, with the highest percentage of homology (42%) being with the 5-HTdro1 receptor, a Drosophila 5-HT receptor positively coupled to adenylate cyclase.

Mouse 5-hydroxytryptamine5A and 5-hydroxytryptamine5B receptors define a new family of serotonin receptors: cloning, functional expression, and chromosomal localization.

Serotonin [5-hydroxytryptamine (5-HT)] is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with guanine nucleotide-binding proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor. Amino acid sequence comparisons revealed that this receptor was a close relative of the previously identified 5-HT5 receptor but was distant from all other 5-HT receptor subtypes; we therefore named it 5-HT5B.

The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5.

Isolation of a mouse "5HT1E-like" serotonin receptor expressed predominantly in hippocampus.

Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors that interact with G proteins, we have isolated from a mouse brain library a cDNA encoding a new serotonin receptor, the 5HT1E beta receptor. Amino acid sequence comparisons revealed that its closest relatives were the recently characterized 5HT1E receptor (S31) and the 5HT1B and 5HT1D receptors. When expressed transiently in Cos-7 cells, the 5HT1E beta receptor displayed a high affinity for the nonspecific serotonergic radioligand 2-[125I]iodolysergic acid diethylamide (Kd = 980 pM).

Mouse 5HT1B serotonin receptor: cloning, functional expression, and localization in motor control centers.

Serotonin is a neuromodulator that mediates a wide range of effects by interacting with multiple receptors. Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated a mouse gene encoding an additional serotonin receptor. When expressed in cultured cells, it displayed the pharmacological profile and coupling with adenylate cyclase characteristic of the 5HT1B receptor subtype.

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems.

Differential expression of the mouse D2 dopamine receptor isoforms.

We have identified and characterized the cDNAs corresponding to the mouse D2 dopamine receptors. We show that in the mouse the D2 dopamine receptor is found in two forms, generated by alternative splicing of the same gene, mRNA distribution analysis of areas expressing the D2 receptors shows that the larger form is the most abundant, except in the brain stem where the shorter form is predominant. Membranes of mammalian cells transiently transfected with both forms of D2 receptor bind [3H]spiperone with a high affinity.

Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.

Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site.

Cloning and characterization of a Drosophila tyramine receptor.

Receptors for biogenic amines such as dopamine, serotonin and epinephrine belong to the family of receptors that interact with G proteins and share a putative seven transmembrane domain structure. Using a strategy based on nucleotide sequence homology between the corresponding genes, we have isolated Drosophila cDNA clones encoding a new member of the G protein-coupled receptor family. This protein exhibits highest homology to the human alpha 2 adrenergic receptors, the human 5HT1A receptor and a recently cloned Drosophila serotonin receptor.

Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary.

The D2-dopamine receptor from bovine anterior pituitary has been purified approximately 33,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized carboxymethyleneoximinospiperone-Sepharose, Datura stramonium lectin-agarose, and hydroxylapatite chromatography. The purification yields a single polypeptide band of Mr approximately 120,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed by labeling with radioiodinated Bolton-Hunter reagent, Coomassie Blue, or silver staining. The purified D2 receptor preparations display a specific activity of approximately 5.

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum.

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific.

Identification and sequence of a binding site peptide of the beta 2-adrenergic receptor.

p-(Bromoacetamido)benzyl-1-[125I]iodocarazolol (125I-pBABC) is a potent derivative of the beta-adrenergic receptor antagonist p-aminobenzylcarazolol. Treatment of the receptor with 125I-pBABC results in efficient covalent incorporation of the ligand into the receptor binding site. Extensive degradation of 125I-pBABC-labeled beta 2-adrenergic receptor with either cyanogen bromide or Staphylococcus aureus V8 protease results in specifically labeled fragments having Mr's of about 1600 and 3500, respectively.

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland.

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.

Binding of [125I]-N-(p-aminophenethyl)spiroperidol to the D-2 dopamine receptor in the neurointermediate lobe of the rat pituitary gland: a thermodynamic study.

The novel iodinated ligand [125I]-N-(p-aminophenethyl)spiroperidol ([125I]NAPS) was used to identify the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland. The binding of [125I]NAPS was of high affinity and saturable, given that the dissociation constant and the maximal binding were 34.7 +/- 4.

Characterization of the radioiodinated analogue of SCH 23390: in vitro and in vivo D-1 dopamine receptor binding studies.

A new radioiodinated molecule, 125I-SCH 38840 (previously referred to as 125I-SCH 23982), has been recently reported to be a D-1 dopamine receptor ligand. The current study confirms and expands the characterization of both the radiolabeled and unlabeled forms of this compound, as well as describing the development of an in vivo D-1 receptor binding assay utilizing the 125I-SCH 38840. The binding of 125I-SCH 38840 to rat striatal membranes, in vitro, was saturable and exhibited a KD of 1.

Biochemical properties of D1 and D2 dopamine receptors.

The physiological action of dopamine are mediated by two distinct subtypes of receptors, D1 and D2 dopamine receptors. D1-receptors are linked to stimulation of adenylate cyclase whereas D2-receptors inhibit the enzyme and may also couple to other signal transduction systems such as ion channels. In order to characterize these receptors at the biochemical level we have developed specific probes for the identification and purification of these proteins.

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein.

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations.

Identification of the binding subunit of the D1-dopamine receptor by photoaffinity crosslinking.

The D1-dopamine receptor from rat striatum has been successfully identified by photoaffinity crosslinking using a newly synthesized radioiodinated derivative of the selective D1-antagonist SCH-23390. This compound, (R,S)-5-(3'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol(SCH-38548), has been radioiodinated by a chloramine T procedure yielding three radioiodinated products. One of these separated congeners (with Rf = 0.

Solubilization and characterization of D2-dopamine receptors in an estrone-induced, prolactin-secreting rat pituitary adenoma.

D2-dopamine (3,4-dihydroxyphenylethylamine) receptors were successfully solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate from an estrone-induced rat pituitary adenoma. Forty-five percent of initial protein and 48% of initial [3H]spiroperidol binding sites were solubilized. The high affinity as well as the stereoselectivity of the sites was preserved.

Identification of the D2-dopamine receptor binding subunit in several mammalian tissues and species by photoaffinity labeling.

Photoaffinity labeling of the D2-dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2-dopaminergic antagonist probe [125I]N-(p-azidophenethyl)spiperone ([125I]N3-NAPS). In tissues containing D2-receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr = 94,000. Specificity of the labeling is typically D2-dopaminergic in character.

Affinity chromatography of the anterior pituitary D2-dopamine receptor.

The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified approximately 1000-fold by affinity chromatography on a new affinity support. This support consists of a (carboxymethylene)oximino derivative of the D2-selective antagonist spiperone (CMOS) covalently attached to Sepharose 4B through a long side chain. The interaction of the solubilized receptor activity with the affinity gel was biospecific.

Synthesis and irreversible beta-adrenergic blockade with a bromoacetamido derivative of betaxolol.

1-[4-(2-Cyclopropylmethoxyethyl)phenoxy]-3-[1-p-(bromoacetamidophe nyl) -2-methyl-2-propylamine]-2-propranol (8), which is a derivative of the beta 1-adrenergic agent betaxolol, was synthesized. Compound 8 showed less potent beta-adrenergic blocking activity than betaxolol in an in vitro test with guinea pig tracheal muscle and left atrium but retained high beta 1-selectivity. Irreversible beta-adrenoceptor antagonism of 8 was assessed by the blockade of the isoproterenol response of the guinea pig atria and by ligand binding studies with rat cerebral cortex.

Photoaffinity labeling of the D2-dopamine receptor using a novel high affinity radioiodinated probe.

The ligand binding subunit of the D2 subtype of the dopamine receptor has been identified by photoaffinity labeling. In order to develop a specific covalent receptor probe, an analogue of the potent D2 selective antagonist spiperone, N-(p-aminophenethyl)spiperone (NAPS) has been synthesized. The aminophenethyl substituent of NAPS can be radioiodinated to theoretical specific radioactivity (2,175 Ci/mmol) and then the arylamine group converted to an arylazide to yield a photosensitive probe [( 125I]N3-NAPS).

A novel radioiodinated high affinity ligand for the D2-dopamine receptor. Characterization of its binding in bovine anterior pituitary membranes.

A novel high affinity dopaminergic ligand, N-(p-aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 Ci/mmol. Binding of this ligand to bovine anterior pituitary membranes is: rapid (40-60 min to equilibrium at 25 degrees C) and reversible (t1/2 = 1 h at 25 degrees C); saturable and of high affinity (KD approximately 20 pM) and displays a typical D2-dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D2 receptor.