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...
Tyramine appears to regulate key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, a Caenorhabditis elegans tyramine receptor, SER-2, was identified that is involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind binding with much lower affinity (K i s of 1.55 ± 0.5 and 1.78 ± 0.6 lM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPcS binding to membranes from cells expressing TYRA-2 (EC 50 of 50 ± 13 nM) and the TA-dependent GTPcS binding is PTX-sensitive suggesting that TYRA-2 may couple to Ga i/o . Based on fluorescence from tyra::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Ga i/o -coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.
Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3 H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical K i values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC 50 values. Tyramine, but not octopamine, also elevates Ca 2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonindependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.
Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D 1 dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D 1 signaling is unclear. We now show that ethanol treatment of D 1 receptor-expressing cells decreases D 1 receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D 1 receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment. Ethanol was also found to modulate specific PKC activities as demonstrated using in vitro kinase assays where ethanol treatment attenuated the activities of lipid-stimulated PKCg and PKCd in membrane fractions, but did not affect the activities of PKCa, PKCb 1 , or PKCe. Importantly, ethanol treatment potentiated D 1 receptormediated DARPP-32 phosphorylation in rat striatal slices, supporting the notion that ethanol enhances D 1 receptor signaling in vivo. These findings suggest that ethanol inhibits the activities of specific PKC isozymes, resulting in decreased D 1 receptor phosphorylation and enhanced dopaminergic signaling.
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