Characterization of novel biogenic amine receptors in the human bloodfluke Schistosoma mansoni by Fouad Sufian El-Shehabi Doctorate of Philosophy Institute of Parasitology McGill University Montreal, Quebec, Canada A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Doctorate of Philosophy August 2009 Fouad El-Shehabi 2009 ABSTRACT The genome of the human bloodfluke Schistosoma mansoni encodes 18 putative biogenic amine-like G-protein-coupled receptors (GPCRs). These receptors are potential targets for the development of antischistosomal drugs. One of these sequences, SmGPR-1 (formerly SmGPCR), was previously cloned and was identified as a histamine receptor. In this study, we expanded the functional analysis of SmGPR-1 by studying its expression and tissue distribution both at the RNA and protein levels in different developmental stages of the parasite. In the second part of the study, we cloned and characterized two structurally related receptors, named SmGPR-2 and SmGPR-3. Bioinformatics analyses showed that the three receptors are members of a new clade of biogenic amine GPCRs and are characterized in part by the absence of a highly conserved aspartate (Asp3.32) of the third transmembrane domain. Like SmGPR-1, our first cloned receptor, SmGPR-2, was activated by histamine and its developmental expression at the mRNA level was similar to that of SmGPR-1, both receptors being upregulated in young schistosomula. However, their tissue localization was different. SmGPR-1 was enriched in the tegument, subtegumental musculature and the suckers, whereas SmGPR-2 was associated with neurons of the subtegumental plexuses. The distribution of these receptors correlated with that of histaminergic neurons, which were also detected in the subtegumental neuronal plexuses, the innervation of the suckers, elements of the central nervous system and transverse commissures. These studies suggest that histamine is an important neurotransmitter system in schistosomes. The third receptor investigated in this study, SmGPR-3, was not responsive to histamine but rather was found to have broad specificity for catecholamines, particularly dopamine and related metabolites. In vitro assays of cultured schistosomula revealed that many of the ligands that interact with SmGPR-3 also have strong effects on larval motility, including dopamine, which caused significant inhibition of movement. The results I suggest that SmGPR-3 is probably one of several catecholamine receptors controlling motor function in these parasites. The pharmacological analysis of SmGPR-2 and SmGPR-3 showed they are atypical histaminergic and dopaminergic receptors, respectively. II ABRÉGÉ Au génome de Schistosoma mansoni, un parasite sanguin de l‟homme, on retrouve 18 récepteurs putatifs à amine biogène couplés aux protéines G (RCPG). Ces récepteurs ont un potentiel thérapeutique contre les infections aux schistosomes. La séquence SmGPR-1 (anciennement SmGPCR) a déjà été clonée et identifiée comme un récepteur à l‟histamine. Une analyse fonctionnelle plus poussée de SmGPR-1 est l‟objet de cette thèse. L‟analyse de taux d‟ARNm et de protéines à différents stades de développement du parasite a servi à l‟étude de l‟expression et la répartition tissulaire de SmGPR-1. Deux récepteurs similaires, de par leur structure, le SmGPR-2 et le SmGPR-3 ont été identifiés, clonés et caractérisés lors de cette étude. Suite à des analyses bioinformatiques, ces trois récepteurs ont révélé leur appartenance à une nouvelle variante de récepteurs à amine biogène couplés aux protéines G caractérisés par l‟absence d‟aspartate conservé (Asp3.32) dans le troisième domaine transmembranaire. Tout comme SmGPR-1, le récepteur SmGPR-2 est activé par l‟histamine, et l‟expression de l‟ARNm est similaire à celle de SmGPR-1, les deux récepteurs étant régulés à la hausse chez les jeunes schistosomes. Toutefois, ils sont localisés à différents endroits, SmGPR-1 se retrouve dans le tégument, la musculature subtégumentaire et les ventouses, tandis que SmGPR-2 est associé aux plexus nerveux subtégumentaires. La localisation de ces récepteurs est similaire à celle des neurones histaminergiques que l‟on retrouve dans les plexus nerveux subtégumentaires, l‟innervation des ventouses, dans certains éléments du système nerveux central et les commissures transversales. Il semblerait que l‟histamine soit un important système neurotransmetteur du schistosome. Le troisième récepteur identifié, SmGPR-3, n‟est pas activé par l‟histamine, mais semble démontrer une spécificité étendue aux catécholamines et tout particulièrement à la dopamine et ses métabolites. Des essais in vitro sur des cultures de jeunes schistosomes ont révélé que plusieurs des ligands qui interagissent avec SmGPR- 3 perturbent considérablement la mobilité des larves, incluant la dopamine qui III provoque une inhibition motrice importante. Ces résultats suggèrent que SmGPR- 3 est probablement un des récepteurs à la catécholamine régulant la fonction motrice de ces parasites. L‟analyse pharmacologique de SmGPR-2 et SmGPR-3 démontre qu‟ils sont respectivement des récepteurs histaminergiques et dopaminergiques atypiques. IV ACKNOWLEDGEMENTS Since I came to this great country as an immigrant, I was searching for universities that contain departments/institutes of Parasitology. I was lucky to get the opportunity to work my research at the Institute of Parasitology of McGill and under the supervision of Professor Paula Ribeiro. Her valuable contributions, suggestions and continuous encouragement, helped me a lot to improve my technical and writing skills. Dr. Ribeiro is a great supervisor with a superb attitude toward her research team. She always listens to her students for any technical problem, giving a superior advice and providing an excellent guidance to resolve it. I would like to say: Thank you Paula very much for your support, excellent recommendations and critiques, especially during the preparation of manuscripts and this dissertation. This thesis would not have been possible without Paula‟s encouragement and support. I really enjoyed my work of Schistosoma mansoni GPCRs project with a knowledgeable scientist like Professor Ribeiro. I would not stop saying thank you Paula for these wonderful years I spent in your lab. I would like to extend my appreciation to all present and past lab-colleagues. Aisha Mousa, Dr. Joseph Nabhan, Serge Dernovici, Nicholas Patocka, Dr. Amira Taman, Laura Sayegh, Dr. Christelle Bouchard, Kristi Bangs, Kevin Macdonald and Dr. Fadi Hamdan. A special thank to Christiane Trudeau, for her excellent assistance in providing French translating of the thesis‟s abstract. During exciting years of this study, I made a friendship with many wonderful people at the Institute of Parasitology. Special thanks to my good friends Dr.Omar Alqawi, Dr. Aws Abdul-Wahid and Ms. Kathy Keller. I also would like to thank Mr. Gorden Bingham, Mrs. Shirley Mongeau, Elizabeth Ruizlancheros, Dr. Manami Nishi, Dr. Cristina Gheorghiu, Yovany Moreno, Alireza Shaneh, Houtan Moshiri, Dr. Mike Osei-Atweneboana, Dr. Jeff Eng, Smriti Kala, Hamed Shaterinajafabadi, Vijayaraghava Rao, Maurice Odiere, Catherine Bourguinat, Sonia Edaye and Dr. Hiren Banerjee. V I also want to show deep appreciation and respect to all the faculty staff at the fascinating institute in the past and current, especially Dr. Timothy Geary, Dr. Roger Prichard, Dr. James Smith, Dr. Marilyn Scott, Dr. Gaetan Faubert, Dr. Reza Salavati, Dr. Robin Beech, Dr. Terry Spithill, Dr. Elias Georges and Dr. Florence Dzierszinski. Finally, I am truly grateful to my dad Sufian and my sisters Basma, Eman and Fida for their unlimited love and encouragement. This thesis is dedicated to the souls of my mother and my grandmother (May ALLAH SWT Bless all of them, Ameen). VI THESIS OFFICE STATEMENT Candidates have the option, subject to the approval of their Department, of including, as part of their thesis, copies of the text of a paper(s) submitted for publication, or the clearly-duplicated text of a published paper(s) provided that these copies are bound as an integral part of the thesis. If this option is chosen, connecting texts, providing logical bridges between the different papers are mandatory. The thesis must still conform to all other requirements of the “Guidelines Concerning Thesis Preparation” and should be in a literary form that is more than a mere collection of manuscripts published or to be published. The thesis must include, as separate chapters or sections: (1) a Table of Contents, (2) a general abstract in English and French, (3) an introduction which clearly states the rationale and objectives of the study, (4) a comprehensive general review of the background literature to the subject of the thesis, when this review is appropriate, and (5) a final overall conclusion and/or summary. Additional material (procedural and design data, as well as descriptions of the equipment used) must be provided where appropriate and in sufficient detail (e.g. in appendices) to allow a clear and precise judgement to be made of the importance and originality of the research reported in the thesis. In the case of manuscripts co-authored by the candidate and others, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent; supervisors must attest to the accuracy of such claims at the Ph.D. Oral defence. Since the task of the examiners is made more difficult in these cases, it is in the candidate‟s interest