Molecular & Biochemical Parasitology 119 (2002) 75 – 86 A novel Schistosoma mansoni G protein-coupled receptor is responsive to histamine  Fadi F. Hamdan a,1 , Mark Abramovitz b , Aisha Mousa a , Jinling Xie a , Yves Durocher c , Paula Ribeiro a, * a Institute of Parasitology, McGill Uniersity, 21,111 Lakeshore Road, Ste. Anne de Belleue, Que., Canada H9X 3V9 b Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, PO Box 1005, Pointe Claire, Doral, Que., Canada H9R 4P8 c NRC Biotechnology Research Institute, 6100 Royalmount Aenue, Montreal, Que., Canada H4P 2R2 Received 8 August 2001; received in revised form 1 October 2001; accepted 2 October 2001 Abstract A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology ( 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn 111 ) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca 2 + (EC 50 =0.54 0.05 M). An Asn 111  Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca 2 + - and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Schistosoma mansoni ; G protein-coupled receptor; Biogenic amine; Histamine; Cloning; Neurotransmitter; Signal transduction www.parasitology-online.com. 1. Introduction Parasitic platyhelminths (flatworms) of the genus Schistosoma are among the most prevalent causes of human parasitic infection and disease. Schistosoma mansoni is a causative agent of schistosomiasis, a debil- itating disease that afflicts millions of people world- wide. Studies of flatworm neurobiology have identified several putative neurotransmitters, among them bio- genic monoamines, which are present within the central nervous system and peripheral tissues of the parasite. The monoamine, serotonin (5-hydroxytryptamine: 5- HT)), has been most extensively studied, and its many functions as a modulator of motility and metabolic regulator have been well documented (reviewed in [1]). In contrast, considerably less is known about the prop- erties of other biogenic amines, in particular histamine. Although better known as a mediator of immunity, histamine also functions as an important neurotrans- mitter in a variety of vertebrate and invertebrate phyla (see [2–4]). Among the flatworms, histamine has been identified in S. mansoni [5] and other trematodes [6], as  Note: Nucleotide Sequence data reported in this paper are avail- able in the GenBank™ data base (SmGPCR: accession number: AF031196). * Corresponding author. Tel.: +1-514-398-7607; fax: +1-514-398- 7857. E-mail address: paula – ribeiro@maclan.mcgill.ca (P. Ribeiro). 1 Present address: Laboratory of Bioorganic Chemistry, Bldg. 8A, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA. 0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0166-6851(01)00400-5