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. The corresponding mRNA is found predominantly in adult Drosophila heads. Membranes from mammalian cells expressing this receptor displayed high affinity binding sites for [3H]yohimbine, an alpha 2 adrenergic receptor antagonist (Kd = 4.45 x 10(‐9) M). Tyramine was the most efficient of the putative Drosophila neurotransmitters at displacing [3H]yohimbine binding (EC50 = 1.25 x 10(‐6) M). Furthermore tyramine induced an inhibition of adenylate cyclase activity in NIH 3T3 cells expressing this receptor. The Drosophila tyramine receptor that we have isolated might therefore be an invertebrate equivalent of the mammalian alpha 2 adrenergic receptors.

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. The 5HT‐dro1 receptor stimulates adenylate cyclase while the 5HT‐dro2A and the 5HT‐dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT‐dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.

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. When stably introduced into mouse NIH 3T3 cell;, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems.Serotonin (5-hydroxytryptamine) is a neurotransmitter found in both vertebrates and invertebrates that plays a role in various physiological mechanisms, including sleep, appetite, pain perception, learning, and the control ofcyclic events (for a review see ref. 1).We decided to study the role of serotonin in Drosophila melanogaster in order to have access to the powerful genetic techniques that are available for this species. In flies, genetic evidence suggests a role of serotonin in learning processes (2). For example, the ddc mutants, which lack the enzyme dopamine decarboxylase and therefore do not synthesize serotonin and dopamine, exhibit altered learning abilities (3). Biochemical and pharmacological studies performed in other insect systems have also suggested a role for serotonin in physiological mechanisms such as salivary gland secretion and the control of circadian rhythms (for a review see ref. 4). The ability of a single neurotransmitter to mediate a wide range of effects is related to the existence of different types of receptors that are coupled to distinct signal-transduction pathways. The recent isolation of three mammalian serotonin receptors has revealed that they belong to the large family of transmembrane proteins that interact with guanine nucleotide-binding proteins (G proteins) (5-7). These G-proteincoupled receptors, which include the muscarinic acetylcholine receptors, the adrenergic receptors, and the opsins, share a predicted seven-transmembrane-domain structure with highly conserved amino acid sequences, especially within certain transmembrane regions (8). The interaction between receptors and various G proteins leads to the activation of different second-messenger pathways. For example, some

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor ␥2 (RAR␥2), RAR␣1, and retinoid X receptor ␣1 (RXR␣1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RAR␥2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXR␣1. In contrast, RAR␣1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXR␣1 degradation is also modulated by heterodimerization. Thus, each partner of RAR␥/RXR␣ and RAR␣/RXR␣ heterodimers modulates the degradation of the other. We conclude that the liganddependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.

Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.

By using RAR type (a, 3B, or

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 nudeotide-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. In NIH 3T3 cells expressig this receptor, serotonin induced a decrease in forskolin-stimulated cAMP levels. This effect was blocked by pertussis toxn, indicating that the 5HT1B receptor interacts with a pertussis toxinsensitive guanine nucleotide-binding protein. To obtain clues as to the possible function of the SHT1B receptor, we have analyzed its pattern of expression in the adult mouse brain by in situ hybridization. Our results, together with previous autoradiographic studies, suggest that the 5HT1B receptors are localized presynaptically on the terminals of striatal neurons and Purkinje cells and that they might modulate the release of neurotransmitters such as y-aminobutyric acid. The predominant expression of the 5HT1B receptor in the striatum and cerebellum points to an involvement of this receptor in motor control.Serotonin (5-HT) is a neuromodulator that is involved in various functions such as sleep, appetite, pain perception, and vascular contraction. This diversity of effects can be related to the fact that the 5-HT-ergic neurons project into virtually all parts of the brain and spinal cord, although their cell bodies are concentrated in a limited area, the raphe nuclei. 5-HT activates multiple receptor subtypes that exhibit distinct pharmacological properties, signaling systems, and tissue distributions (for a review, see ref. 1). The 5HT1B receptors have been identified in the rat and mouse brain where their highest density was found within the globus pallidus and the substantia nigra. However, they could not be detected in the brain of other species, including humans. These species contained, instead, 5HT1D receptors that have a slightly different pharmacological profile but the same tissue distribution. It was therefore suggested that the 5HT1B and iD receptors correspond to species variants of a same receptor subtype. The 5HT1B and the 5HT1D receptors are negatively coupled with adenylate cyclase. Recent cloning of the 5HT1A, 5HT1C, and 5HT2 receptors has revealed that they belong to the large family of receptors that interact with guanine nucleotide-binding proteins (G proteins) and share a predicted seven-transmembrane-domain structure (2). We have exploited the sequence homologies that exist between several members of this family to clone the gene encoding the mouse 5HT1B receptor. One microgram of DNA was annealed at 550C and amplified at 720C in the presence of 3 mM MgCl2 for 20 cycles with primers i and ii and for 20 more cycles with primers i and iii. The PCR products were cut with Xho I and Kpn I cloned in...

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. When expressed in Cos‐7 cells and NIH‐3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5‐HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.