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.