92 The Journal of Experimental Biology 211, 92-105 Published by The Company of Biologists 2008 doi:10.1242/jeb.012450 Conservation of structure, signaling and pharmacology between two serotonin receptor subtypes from decapod crustaceans, Panulirus interruptus and Procambarus clarkii Nadja Spitzer*, Donald H. Edwards and Deborah J. Baro† Department of Biology, Georgia State University, Atlanta, GA 30302, USA *Present address: 219 BBSC, Department of Biology, College of Science, Marshall University, 1 John Marshall Drive, Huntington, WV 25755, USA †Author for correspondence (e-mail: [email protected]) Accepted 25 October 2007 SUMMARY Serotonin (5-HT) plays important roles in the maintenance and modulation of neural systems throughout the animal kingdom. The actions of 5-HT have been well characterized for several crustacean model circuits; however, a dissection of the serotonergic transduction cascades operating in these models has been hampered by the lack of pharmacological tools for invertebrate receptors. Here we provide pharmacological profiles for two 5-HT receptors from the swamp crayfish, Procambarus clarkii: 5-HT2␤ and 5-HT1␣. In so doing, we also report the first functional expression of a crustacean 5-HT1 receptor, and show that it inhibits accumulation of cAMP. The drugs mCPP and quipazine are 5-HT1␣ agonists and are ineffective at 5-HT2␤. Conversely, methiothepin and cinanserin are antagonists of 5-HT2␤ but do not block 5-HT1␣. A comparison of these two receptors with their orthologs from the California spiny lobster, Panulirus interruptus, indicates conservation of protein structure, signaling and pharmacology. This conservation extends beyond crustacean infraorders. The signature residues that form the ligand-binding pocket in mammalian 5-HT receptors are found in the crustacean receptors. Similarly, the protein domains involved in G protein coupling are conserved between the two crustacean receptors and other characterized arthropod and mammalian 5-HT receptors. Considering the apparent conservation of pharmacological properties between crustacean 5-HT receptors, these tools could be applicable to related crustacean physiological preparations. Key words: agonist, antagonist, neuromodulation, G protein-coupled receptor, amine, cloning. INTRODUCTION of these receptors might change in response to social or Serotonin (5-HT) is an important neurotransmitter and environmental stimuli. neurohormone in most animal species. Diverse processes, including Crustacean hormonal circuits with small numbers of identifiable social behaviors and a variety of systemic physiological functions cells are ideal preparations for investigating the mechanistic basis are regulated by this biogenic amine. Animals have evolved a suite of modulation and plasticity. Such studies have, however, been of 5-HT receptor types; thus, a single neurotransmitter can have limited by the lack of pharmacological tools. Although the protein multiple and complex effects on different tissues. To date, seven sequences and second-messenger couplings of 5-HT receptors are classes of serotonin receptors have been identified in mammals. relatively well conserved across all species, their pharmacological These receptor classes are categorized with respect to their signal profiles can vary significantly between vertebrate and invertebrate transduction mechanisms and pharmacological properties preparations, and even within invertebrate preparations (Tierney, (Gerhardt and van Heerikhuizen, 1997; Hoyer et al., 2002; Kroeze 2001). This is not surprising as one would not expect selection for et al., 2002). or against amino acids involved in binding synthetic ligands. We In invertebrate nervous systems 5-HT can modulate motor have recently identified and cloned two crustacean 5-HT receptors pattern generation (Hooper and DiCaprio, 2004), escape and social from the spiny lobster, Panulirus interruptus, infraorder Achelata: status (Edwards et al., 1999), aggression (Kravitz, 2000) and 5-HT1␣Pan and 5-HT2␤Pan (Clark et al., 2004; Sosa et al., 2004). We learning (Barbas et al., 2003; Bicker, 1999; Kandel and Schwartz, were interested to know whether the actions of pharmacological 1982). Serotonin has multiple and complex roles in crustacean agents were conserved across crustacean species. We therefore models. 5-HT application to the stomatogastric nervous system of obtained full-length clones for the same two receptors from a decapod crustaceans elicits distinct responses from individual second distantly related decapod crustacean, the swamp crayfish, identified neurons (Flamm and Harris-Warrick, 1986), and these Procambarus clarkii (infraorder Astacidea), and expressed these responses can differ for the same identified neuron in different receptors in a heterologous system in order to determine their species (Katz and Tazaki, 1992). In the crayfish, the social status second messenger couplings and pharmacological profiles. These of an individual determines the directionality of 5-HT modulation parameters were then compared across the two species. of the lateral giant escape circuit response to sensory stimulation (Yeh et al., 1997; Yeh et al., 1996). The rate and concentration of MATERIALS AND METHODS 5-HT application to the circuit also affect this response (Teshiba et Animals al., 2001). These studies suggest that crustaceans express several Crayfish, Procambarus clarkii Girard 1852, were obtained from different 5-HT receptor types and that the expression or signaling Atchafalaya Biological Supply (Raceland, LA, USA) and kept Conservation of crustacean 5-HT receptors 93 communally at 20°C in 40·l tanks with continual filtration and Sequence data were analyzed using Sequencher 4.1 (Gene Codes aeration. Corp., Ann Arbor, MI, USA). Sequences for other arthropod species were obtained from GenBank and alignments were created Chemicals and cell lines to determine sequence identities using the ClustalW algorithm with HEK293, NIH3T3, COS-7 and MDCK cells, Earle’s minimal default settings in Lasergene MegAlign (DNASTAR Inc., essential medium (EMEM), horse serum, trypsin, penicillin and Madison, WI, USA). Full Procambarus sequences have been streptomycin were obtained from the American Type Culture deposited in GenBank under accession numbers EU131666 (5- Collection (Mannassas, VA, USA). Dulbecco’s modified Eagle’s HT2␤Pro) and EU131667 (5-HT1␣Pro). medium (DMEM) was from Mediatech Inc. (Herndon, VA, USA). Dialyzed fetal bovine serum (FBS), TRex cell line (293-TR), Generation of cell cultures expressing crustacean 5-HT pDNA4/TO plasmid, blasticidin and zeocin were from Invitrogen receptors (Carlsbad, CA, USA). Cinanserin was obtained from Tocris We previously characterized the signaling and pharmacological (Ballwin, MO, USA). All other chemicals were from Sigma (St properties of the 5-HT2␤Pan receptor transiently expressed in Louis, MO, USA). For pharmacology experiments, amine and HEK293 cells (Clark et al., 2004). We used the same techniques to –1 –1 agonist stock solutions (10 ·mol·l ) were made fresh for every characterize the Procambarus ortholog, 5-HT2␤Pro. Briefly, cells experiment in medium or 50% ethanol, respectively. Two were maintained in EMEM supplemented with 10% FBS, exceptions were tyramine (Tyr), which was made fresh as a 50·i.u.·ml–1 penicillin and 50·␮g·ml–1 streptomycin (normal 10–2·mol·l–1 stock in medium, and methysergide, which was made medium). Cells were plated on 60·mm dishes in EMEM without as a 10–2·mol·l–1 stock in DMSO and stored at –20°C. Antagonist antibiotics, allowed to grow to 95–100% confluency and then drugs were made as 10–2·mol·l–1 stock solutions in DMSO and transfected with 2·␮g of DNA using lipofectamine (Invitrogen). stored at –20°C. The plates were supplemented to a final concentration of 10% FBS 6·h after transfection, and the medium was replaced with normal Cloning of full-length 5-HT1␣ and 5-HT2␤ from Procambarus medium 24·h after transfection. clarkii and generation of expression constructs In order to functionally characterize 5-HT1␣Pan and 5-HT1␣Pro, Complete cloning and sequencing of 5-HT2␤Pan and 5-HT1␣Pan from we first generated full-length constructs using standard Panulirus interruptus have been previously described (Clark et al., recombinant techniques, as described above. The 5-HT1␣ receptors 2004; Sosa et al., 2004). were first cloned into pIRESneo and stably transfected into several We also previously cloned a large segment of 5-HT1␣Pro spanning cell lines using lipofectamine as detailed in the Results. transmembrane domains III–VII from Procambarus clarkii (Sosa et Immunoblotting experiments coupled with the lack of growth after al., 2004). We have now completed the sequencing of the 5-HT1␣Pro several weeks of selection suggested that none of these cell lines cDNA using rapid amplification of DNA ends (SMART RACE could stably express either the 5-HT1␣Pan or the 5-HT1␣Pro receptor. cDNA Amplification kit; BD Biosciences, Clontech, Cambridge, This did not appear to be a general phenomenon associated with UK) as previously described (Clark et al., 2004). Constructs crustacean receptors, as we successfully expressed 5HT2␤ receptors containing the complete ORF were assembled using standard and crustacean dopamine receptors in these cell lines. The most procedures (Ausubel et al., 1990). Both strands of the construct were reasonable explanation for our finding was that the cell lines could sequenced and errors that had been introduced in the cloning process not tolerate high levels of the 5-HT1␣ receptor, and for reasons were