Journal of Neurochemistry, 2005, 92, 375–387 doi:10.1111/j.1471-4159.2004.02867.x Mutagenesis analysis of the serotonin 5-HT2C receptor and a Caenorhabditis elegans 5-HT2 homologue: conserved residues of helix 4 and helix 7 contribute to agonist-dependent activation of 5-HT2 receptors Jinling Xie,1 Serghei Dernovici and Paula Ribeiro Institute of Parasitology, McGill University, Ste Anne de Bellevue, Quebec, Canada Abstract agonist binding affinity and significantly lower constitutive An alignment of serotonin [5-hydroxytryptamine (5-HT)] G pro- activity compared with wildtype. Mutagenesis of S7.45 in the tein-coupled receptors identified a lysine at position 4.45 (helix C. elegans receptor increased serotonin binding affinity by up to 4) and a small polar residue (serine or cysteine) at 7.45 (helix 7) 25-fold and decreased Emax by up to 65%. The same mutations that occur exclusively in the 5-HT2 receptor family. Other of the cognate C7.45 in rat 5-HT2C produced a smaller fourfold serotonin receptors have a hydrophobic amino acid, typically a change in the affinity for serotonin and decreased agonist methionine, at 4.45 and an invariant asparagine at 7.45. The efficacy by up to 50%. Substitutions of S/C7.45 did not produce functional significance of these class-specific substitutions was a significant change in the basal activity of either receptor. All tested by site-directed mutagenesis of two distantly related mutants tested exhibited levels of receptor expression similar 5-HT2 receptors, Caenorhabditis elegans 5-HT2ce and rat to the corresponding wildtype based on measurements of 5-HT2C. Residues 4.45 and 7.45 were each mutated to a specific [3H]-mesulergine binding or flow cytometry analyses. methionine and asparagine, respectively, or an alanine and the Taken together, these results suggest that K4.45 and S/C7.45 resulting constructs were tested for activity. A K4.45M mutation play an important role in the conformational rearrangements decreased serotonin-dependent activity (Emax) of the rat leading to agonist-induced activation of 5-HT2 receptors. 5-HT2C receptor by 60% and that of the C. elegans homologue Keywords: Caenorhabditis elegans, G protein-coupled by 40%, as determined by a fluorometric plate-based calcium receptor, 5-HT2C, mutagenesis, serotonin. assay. The rat mutant also exhibited nearly sixfold higher J. Neurochem. (2004) 92, 375–387. Serotonin [5-hydroxytryptamine (5-HT)] is a ubiquitous classes of 5-HT receptors belong to the large superfamily of neuroactive agent of both vertebrates and invertebrates. In seven transmembrane-spanning G protein-coupled receptors mammals, 5-HT regulates a variety of physiological phe- (GPCR). nomena in the CNS and periphery, including cognition, As a group, 5-HT2 receptors are characterized by having a sleep, pain perception, mood, feeding behavior, sexual relatively lower affinity for indolealkylamines, including behavior, temperature regulation and gastrointestinal func- tion (Weiger 1997). Among invertebrates, 5-HT acts as both a neurotransmitter and hormone and mediates feeding, Received May 19, 2004; revised manuscript received September 3, locomotion, circadian rhythm, defense behavior and meta- 2004; accepted September 10, 2004. bolic activity across various invertebrate phyla (Walker et al. Address correspondence and reprint requests to Paula Ribeiro, Insti- tute of Parasitology, McGill University, 21 111 Lakeshore Road, Ste 1996). This diversity of effects is mediated by multiple 5-HT Anne de Bellevue, Quebec, Canada H9X 3V9. receptors, a total of seven structurally distinct receptor E-mail: [email protected] classes (5-HT1–7), each of which is further divided into 1The present address of Jinling Xie is Vanderbilt University Medical several subtypes (Boess and Martin 1994). With the excep- Center, Department of Pharmacology, 452 Preston Research Building, tion of the mammalian 5-HT3 ionotropic receptor and a 23rd Avenue South at Pierce, Nashville, TN 37232-6600, USA. Abbreviations used: FACS, fluorescence-activated cell sorting; GPCR, recently identified nematode (roundworm) 5-HT-gated chlor- G protein-coupled receptor; 5-HT, 5-hydroxytryptamine; 5-HT2ce, Ca- ide channel (Ranganathan et al. 2000) all other known enorhabditis elegans 5-HT2 receptor; TM, transmembrane domain. Ó 2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387 375 376 J. Xie et al. serotonin itself, and are preferentially linked to the Gq/phosp- pCIneo and includes the complete coding sequence of 5-HT2ce fused holipase C-b pathway of signal transduction. Three 5-HT2 at the C-terminal end to a FLAG epitope (Hamdan et al. 1999) For subtypes have been identified in mammals, 5-HT2A, 2B and studies of rat 5-HT2C, a cDNA encoding the full-length unedited 2C, which differ on the basis of primary structure and receptor was obtained from the American Type Culture Collection pharmacological profiles (Roth et al. 1998). In addition, (ATCC, Manassas, VA, USA) and modified by PCR to introduce an N-terminal FLAG epitope. The resulting construct was subcloned 5-HT2 receptors have been cloned from invertebrates, including between the NheI/NotI sites of pCEP4 mammalian expression vector Drosophila (Colas et al. 1995), the snail Lymnaea (Gerhardt (Invitrogen, Burlington, Canada), confirmed by DNA sequencing and et al. 1996) and two nematodes (roundworms), Caenorhabditis used for site-directed mutagenesis. All point mutations were generated elegans (Hamdan et al. 1999) and the pig parasite Ascaris suum with the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, (Huang et al. 1999, 2002). The Drosophila and Lymnaea USA), according to the recommendations of the manufacturer. The receptors show some binding characteristics of the mammalian mutations were verified by sequencing the full-length cDNAs. 5-HT2B prototype, whereas the nematode receptors have a distinctive pharmacological profile and may constitute a Cell culture and transfection separate subtype of 5-HT2 receptor (Hamdan et al.1999). COS7 were grown in Dulbecco’s modified Eagle’s medium The finding of 5-HT2 receptors in the lower invertebrates supplemented with 10% bovine fetal serum (Invitrogen) and supports the notion that the 5-HT2 class diverged early in 20 mM HEPES buffer at 37°C in a humidified environment containing 5% CO . HEK293(EBNA1) cells were cultured in evolution, at least before the separation of nematodes. 2 Dulbecco’s modified Eagle’s medium containing L-glutamine and An impressive amount of research over the last few years has supplemented with 10% fetal bovine serum (Invitrogen), 1 mM been aimed at unveiling the structural organization of the sodium pyruvate, 250 lg/mL G418 and 20 mM HEPES buffer 5-HT2 receptor, particularly the 5-HT2A and 2C subtypes. In (Invitrogen). For transfection, cells were seeded in HEPES-buffered the absence of high-resolution crystallographic data, which are Dulbecco’s modified Eagle’s medium containing 10% dialysed fetal still lacking for all biogenic amine GPCRs, most of the bovine serum and cultured overnight to approximately 80% information available on 5-HT2 structure is derived from confluency. Unless indicated otherwise, cells were transfected in mutagenesis analyses and comparisons with bovine rhodopsin, 100-mm culture dishes (1.5–2.5 · 106 cells and 3 lg plasmid DNA/ the class I GPCR prototype (Palczewski et al. 2000; Teller dish) using FuGENE 6 (Roche Diagnostics, Laval, Canada), et al. 2001). This research has identified a number of primary according to the specifications of the manufacturer. Transfection agonist binding residues located mainly on transmembrane efficiency was monitored routinely by using a green fluorescence protein-encoding plasmid (pTracer) and was typically 40–50%. domains (TM) 3, 5 and 6, which are believed to constitute a core of the receptor’s binding pocket (Choudhary et al. 1995; Binding assays Almaula et al. 1996; Roth et al. 1997; Kristiansen et al. 2000; Binding assays of 5-HT2ce wildtype and mutants were performed Shapiro et al. 2000; Visier et al. 2002a; Ebersole et al. 2003). on crude membrane preparations of transiently transfected COS7 A number of other residues identified on TM2, 3, 6 and 7 and cells. Rat 5-HT2C wildtype and mutants were transiently expressed intracellular loops have been implicated in receptor activation in HEK293(EBNA1) cells. In both cases, transiently transfected and G protein coupling (Sealfon et al. 1995; Herrick-Davis cells were harvested 48 h post-transfection and lysed by brief et al. 1997, 1999; Roth et al. 1997; Prioleau et al. 2002; sonication in ice-cold TEM buffer (50 mM Tris-HCl, pH 7.4, Shapiro et al. 2002; Visiers et al. 2002a,b) Despite these 0.5 mM EDTA, 10 mM MgCl2). Binding assays were performed advances, however, a great deal remains to be learned about the with aliquots (5–10 lg protein/reaction) of a 28 000 g crude structural organization of serotonergic GPCRs, in particular the membrane preparation in a total volume of 200 lL TEM buffer containing [3H]-mesulergine (75–86 Ci/mmol; Amersham, Baie subtle differences between the various receptor subtypes. In d’Urfe´, Canada) as the radiolabeled ligand. Saturation curves were this study, we have used site-directed mutagenesis to test a generated from a minimum of seven different labeled ligand number of TM4 and 7 residues which were found to occur concentrations in the presence and absence of 10 lM mianserin exclusively in 5-HT2 receptors, both vertebrate and inverteb- (Sigma, Oakville, Canada) for measurements of non-specific rate, and thus were postulated to have functional significance. A binding. Competition studies were performed by testing seven to mutagenesis analysis of two distantly related 5-HT2 receptors, eight concentrations of unlabeled competitor in the presence of a rat 5-HT2C and C. elegans 5-HT2ce, implicated a conserved constant amount of [3H]-mesulergine. All test ligands were prepared lysine of TM4 (K4.45) and a small polar residue of TM7 (S or in 0.1% ascorbic acid. Reaction mixtures were incubated at room C7.45) in the conformational activation of both receptors.