Characterization of a Novel Serotonin Receptor from Caenorhabditis elegans: Cloning and Expression of Two Splice Variants Fadi F. Hamdan, *Mark D. Ungrin, *Mark Abramovitz, and Paula Ribeiro Institute of Parasitology, McGill University, Ste. Anne de Bellevue; and *Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada Abstract: Serotonin [5-hydroxytryptamine (5-HT)] modu- lates feeding activity, egg-laying, and mating behavior in the free-living nematode, Caenorhabditis elegans. We have cloned a novel receptor cDNA from C. elegans (5-HT 2Ce ) that has high sequence homology with 5-HT 2 receptors from other species. When transiently ex- pressed in COS-7 cells, 5-HT 2Ce exhibited 5-HT binding activity and activated Ca 2+ -mediated signaling in a man- ner analogous to other 5-HT 2 receptors. However, 5-HT 2Ce displayed unusual pharmacological properties, which resembled both 5-HT 2 and 5-HT 1 -like receptors but did not correlate well with any of the known 5-HT 2 subtypes. Two splice variants of 5-HT 2Ce that differ by 48 N-terminal amino acids were identified. The two isoforms were found to have virtually identical binding and signal- ing properties but differed in their levels of mRNA expres- sion, with the longer variant being four times more abun- dant than the shorter species in all developmental stages tested. Taken together, the results describe two variants of a novel C. elegans 5-HT receptor, which has some of the properties of the 5-HT 2 family but whose pharmaco- logical profile does not conform to any known class of receptor. Key Words: Caenorhabditis elegans—Seroto- nin—5-HT 2 receptor—G protein-coupled receptor— Cloning—Expression—Spliced leader—Aequorin. J. Neurochem. 72, 1372–1383 (1999). Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neuroactive agent of vertebrates and inverte- brates. In the free-living nematode Caenorhabditis el- egans, 5-HT has been identified within several central and peripheral neurons, including the well-characterized pharyngeal neurosecretory motorneurons, the hermaph- rodite-specific neurons, and the male-specific CP neu- rons (Horvitz et al., 1982; Desai et al., 1988; Loer and Kenyon, 1993). The serotonergic neurosecretory motor neurons may modulate pharyngeal pumping (feeding), locomotion, and egg laying, whereas the hermaphrodite- specific neurons and CP neurons seem to affect egg- laying and male mating behavior, respectively (Horvitz et al., 1982; Desai and Horvitz, 1989; Loer and Kenyon, 1993). These effects of 5-HT are mediated, in part, by G proteins (Bargmann and Kaplan, 1998) and at least one type of G protein-coupled receptor (GPCR), which is negatively linked to adenylate cyclase (Olde and Mc- Combie, 1997). It is unknown at present if there are other receptors and pathways of signal transduction that me- diate the multiple effects of 5-HT in this animal. In mammals, where 5-HT is a well-established neuro- transmitter involved in a wide range of physiological activities (Leonard, 1994), as many as seven different classes of 5-HT receptors (5-HT 1 –5-HT 7 ) have been identified, all of which are further divided into multiple subtypes (Hoyer et al., 1994; Gerhardt and Van Heer- ikhuizen, 1997). The vast majority of mammalian 5-HT receptors belong to the large GPCR superfamily and couple to adenylate cyclase, either positively (5-HT 4 , 5-HT 6 , and 5-HT 7 ) or negatively (5-HT 1 ). In contrast, the 5-HT 2 family has very distinctive structural properties and couples to phospholipase C and the phosphoinositol (inositol trisphosphate)/Ca 2+ -mediated pathway of sig- nal transduction (Hoyer et al., 1994; Gerhardt and Van Heerikhuizen, 1997). Received October 15, 1998; revised manuscript received November 30, 1998; accepted December 7, 1998. Address correspondence and reprint requests to Dr. P. Ribeiro at Institute of Parasitology, McGill University, Macdonald Campus, 21, 111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9. Abbreviations used: CeIF, Caenorhabditis elegans eukaryotic initi- ation factor 4A homologue; DOI, 2,5-dimethoxy-4-iodoamphetamine; GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine (sero- tonin); 5-HT Asc , putative 5-hydroxytryptamine 2 -like sequence recently cloned from the parasitic nematode Ascaris suum; 5-HT 2Ce , 5-hydroxy- tryptamine 2 receptor from Caenorhabditis elegans; 5-HT 2CeL and 5-HT 2CeS , long and short, respectively, 5-hydroxytryptamine 2 receptor isoform from Caenorhabditis elegans; 5-HT 2Dro , 5-hydroxytrypta- mine 2 receptor cloned from Drosophila; 5-HT 2Lym , 5-hydroxytrypta- mine 2 receptor cloned from the pond snail, Lymnaea; LSD, lysergic acid diethylamide; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetra- lin; ORF, open reading frame; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; SL, spliced leader; TEM buffer, 50 mM Tris (pH 7.4), 0.5 mM EDTA, and 10 mM MgCl 2 ; TM, transmem- brane domain; UTR, untranslated region. 1372 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry