Identification of Conserved Aromatic Residues Essential for Agonist Binding and Second Messenger Production at 5-Hydroxytryptamine<sub>2A</sub>Receptors

Roth, Bryan L.; Shoham, Menachem; Choudhary, M S; Khan, Naseem

doi:10.1124/mol.52.2.259

Cited by 131 publications

(143 citation statements)

References 24 publications

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“…Ser (Strader et al 1989;Choudhary et al 1993;Almaula et al 1996;Roth et al 1997), G-protein coupling (DRYXXIXXPI153) (Moro et al 1993) and desensitization/internalization (NPXXY412) (Barak et al 1994) were conserved in both SER-2 and SER-2a (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a tyramine receptor from Caenorhabditis elegans

Rex

Komuniecki

2002

Journal of Neurochemistry

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Octopamine (OA) plays an important role in the regulation of a number of key processes in nematodes, including pharyngeal pumping, locomotion and egg-laying. However, while putative OA receptors can be tentatively identified in the Caenorhabditis elegans database, no OA receptors have been functionally characterized from any nematode. We have isolated two cDNAs, ser-2 and ser-2a, encoding putative C. elegans serotonin/OA receptors (C02D4.2, ser-2). The sequences of these cDNAs differ from that predicted by GeneFinder and lack 42 bp of exon 2. In addition, ser-2a appears to be alternatively spliced and lacks a predicted 23 amino acids in the third intracellular loop. COS-7 cells expressing SER-2 bind [ 3 H]LSD in the low nM range and exhibit K i s for tyramine, octopamine and serotonin of 0.07, 2, and 13.7 lM, respectively. Significantly, tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells stably expressing SER-2 with an IC 50 of about 360 nM, suggesting that SER-2 is a tyramine receptor. Keywords: Caenorhabditis elegans, cyclic AMP, G-protein coupled receptor, tyramine. Understanding the signaling pathways involved in key nematode-specific processes, such as locomotion, pharyngeal pumping and egg laying, may aid in the identification of novel targets for the development of new classes of anthelmintics. The biogenic amine octopamine (OA) is a neuroactive ligand in many invertebrates and appears to play an important role in nematode physiology. The biosynthesis of OA requires the hydroxylation of tyramine (TA) to OA by tyramine b-hydroxylase and the effects of OA and TA are mediated by G-protein coupled receptors, many of which positively or negatively modulate adenylyl cyclase activity (Roeder 1999). Studies on the physiological role and signaling of OA and its precursor TA have focused largely on insects where, OA and TA regulate a variety of processes, including energy generation via fat body metabolism (Downer 1979;Wang et al. 1990;Blau and Wegener 1994;Park and Keeley 1998), regulation of egg-laying (Monastirioti et al. 1996;Torfs et al. 2000) and induction of feeding (Braun and Bicker 1992). The role of TA and OA in mammals is not well studied but has recently gained much attention due to the isolation of human and rodent receptors that specifically bind TA and couple to G as , highlighting the need to re-evaluate the role of these trace amines in vertebrates (Borowsky et al. 2001).In the free-living nematode, Caenorhabditits elegans, a putative tyramine-b hydroxylase (tbh-1) has been identified and localized to the two RIC interneurons, suggesting they are octopaminergic (Alkema and Horvitz, personal communication). Mutants lacking tbh-1 move more slowly than wild type, indicating a role for OA in locomotion. Indeed, ectopic application of OA to wild type C. elegans enhances locomotion and inhibits pharyngeal pumping and egg laying (Horvitz et al. 1982). Significantly, the inhibition of pharyngeal pumping induced by OA in C. elegans appears to be mediated by G ao . OA inhibits the fir...

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Section: Resultsmentioning

confidence: 99%

Characterization of a tyramine receptor from Caenorhabditis elegans

Rex

Komuniecki

2002

Journal of Neurochemistry

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“…Various aromatic amino acids in the fourth and other TM domains have been studied for ligand binding and signaling in a large variety of GPCRs, including the m 1 and m 3 muscarinic, 5-HT 2 serotonergic, NK 2 tachykinin, and ␦-opioid receptors (Wess et al, 1992(Wess et al, , 1993Choudhary et al, 1993;Matsui et al, 1995;Befort et al, 1996;Roth et al, 1997;Renzetti et al, 1999a,b). In similarity to the data presented here, Befort et al (1996) found that W173 of the ␦-opioid receptor (in an analogous position to W158 of CB 2 ) is involved in receptor- ligand recognition and that mutation of this amino acid reduced the receptor's binding affinity to many ␦ ligands.…”

Section: Discussionmentioning

confidence: 99%

Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB₂ Cannabinoid Receptor

Rhee

Nevo

Bayewitch

et al. 2000

Journal of Neurochemistry

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Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB 2 cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB 1 and CB 2 cannabinoid receptors but not in many other GPCRs) of the human CB 2 receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB 2 receptor.

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“…f14d12.6(ok371) has a 1072 bp deletion that removes a portion of exons I-VI and predicted TMs I-V, whereas tyra-3(ok325) has a 781 bp deletion that removes portions of exons IV-V and TMs III-V. Therefore, both f14d12.6(ok371) and tyra-3(ok325) are predicted null alleles, because these TMs are essential for ligand binding (Strader et al, 1989;Choudhary et al, 1993;Almaula et al, 1996;Roth et al, 1997). Under standard conditions, tyra-3(ok325) and f14d12.6(ok371) animals exhibited no gross behavioral defects with respect to locomotion, feeding, or egg retention.…”

Section: Resultsmentioning

confidence: 99%

Tyramine and Octopamine Independently Inhibit Serotonin-Stimulated Aversive Behaviors inCaenorhabditis elegansthrough Two Novel Amine Receptors

Wragg¹,

Hapiak²,

Miller³

et al. 2007

J. Neurosci.

114

146

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Biogenic amines modulate key behaviors in both vertebrates and invertebrates. In Caenorhabditis elegans, tyramine (TA) and octopamine (OA) inhibit aversive responses to 100%, but not dilute (30%) octanol. TA and OA also abolish food-and serotonin-dependent increases in responses to dilute octanol in wild-type but not tyra-3(ok325) and f14d12.6(ok371) null animals, respectively, suggesting that TA and OA modulated responses to dilute octanol are mediated by separate, previously uncharacterized, G-protein-coupled receptors. TA and OA are high-affinity ligands for TYRA-3 and F14D12.6, respectively, based on their pharmacological characterization after heterologous expression. f14d12.6::gfp is expressed in the ASHs, the neurons responsible for sensitivity to dilute octanol, and the sra-6-dependent expression of F14D12.6 in the ASHs is sufficient to rescue OA sensitivity in f14d12.6(ok371) null animals. In contrast, tyra-3::gfp appears not to be expressed in the ASHs, but instead in other neurons, including the dopaminergic CEP/ADEs. However, although dopamine (DA) also inhibits 5-HT-dependent responses to dilute octanol, TA still inhibits in dop-2; dop-1; dop-3 animals that do not respond to DA and cat-2(tm346) and Pdat-1::ICE animals that lack significant dopaminergic signaling, suggesting that DA is not an intermediate in TA inhibition. Finally, responses to TA and OA selectively desensitize after preexposure to the amines. Our data suggest that although tyraminergic and octopaminergic signaling yield identical phenotypes in these olfactory assays, they act independently through distinct receptors to modulate the ASH-mediated locomotory circuit and that C. elegans is a useful model to study the aminergic modulation of sensory-mediated locomotory behaviors.

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Identification of Conserved Aromatic Residues Essential for Agonist Binding and Second Messenger Production at 5-Hydroxytryptamine_2AReceptors

Cited by 131 publications

References 24 publications

Characterization of a tyramine receptor from Caenorhabditis elegans

Characterization of a tyramine receptor from Caenorhabditis elegans

Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB₂ Cannabinoid Receptor

Tyramine and Octopamine Independently Inhibit Serotonin-Stimulated Aversive Behaviors inCaenorhabditis elegansthrough Two Novel Amine Receptors

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Identification of Conserved Aromatic Residues Essential for Agonist Binding and Second Messenger Production at 5-Hydroxytryptamine2AReceptors

Cited by 131 publications

References 24 publications

Characterization of a tyramine receptor from Caenorhabditis elegans

Characterization of a tyramine receptor from Caenorhabditis elegans

Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB2 Cannabinoid Receptor

Tyramine and Octopamine Independently Inhibit Serotonin-Stimulated Aversive Behaviors inCaenorhabditis elegansthrough Two Novel Amine Receptors

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Identification of Conserved Aromatic Residues Essential for Agonist Binding and Second Messenger Production at 5-Hydroxytryptamine_2AReceptors

Functional Role of Tryptophan Residues in the Fourth Transmembrane Domainof the CB₂ Cannabinoid Receptor