Alternative-splicing of serotonin receptor isoforms in the pharynx and muscle of the parasitic nematode, Ascaris suum.
Pharyngeal pumping is essential for nematode feeding and survival and is dramatically stimulated by serotonin (5-HT). In the present study, a cDNA pool was prepared from poly A + RNA isolated from pharynxes dissected from the parasitic nematode, Ascaris suum, and was used as a template for RT-PCR with degenerate primers designed from sequences conserved in 5-HT receptors from a variety of sources. A putative 5-HT receptor cDNA (AS1) was identified which exhibited most identity to the 5-HT2 family of receptors. AS1 was 1925 nucleotides, did not appear to be trans-spliced and contained a 3' untranslated region of 127 nucleotides with a polyadenylation signal (ATTAAA) and a short poly A+ tail. The coding region predicted a protein of 532 amino acids with a molecular weight of 60 176. When AS1 was transiently expressed in COS-7 cells, isolated membranes exhibited the high affinity, saturable binding of [125I]LSD. More importantly, [125I]LSD binding was inhibited by 5-HT, but not other biogenic amines, supporting the identification of AS1 as a 5-HT receptor. Additional cDNAs identical, in part, to AS1 were also identified. AS1deltaIV lacked a predicted 42 amino acids at the carboxy terminus of the third intracellular loop, while AS2 and AS3 contained different COOH-termini, regions implicated in G-protein coupling in other heptahelical receptors. A portion of the gene (5htn) encoding AS1 also was cloned and sequenced. This genomic fragment was about 10 kb, contained the entire AS1 open reading frame and included eight exons and seven introns. From this analysis, it appears that these different AS cDNAs were generated by alternative-splicing, AS1deltaIV from the deletion of exon IV, and AS2 and AS3 from the use of alternative sites within exon VII as 5' splice acceptor sites for exon VIII. Using RT-PCR and primers specific for each of the isoforms, AS1 -3 appeared to be expressed in pharynx, while only AS1 and AS2 were present in body wall muscle. More importantly, the deletion of exon IV appeared to be associated exclusively with AS1 in pharynx and AS2 in muscle.
Department of Biology, University of Toledo, OH 43606, USA. xhuang4@uoft02.utoledo.edu
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Huang
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Duran
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Diaz
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Xiao
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Messer
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Komuniecki
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D008238:Lysergic Acid Diethylamide / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009928:Organ Specificity; D010614:Pharynx / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D018509:DNA, Helminth; D020033:Protein Isoforms; D011985:Receptors, Serotonin; D008238:Lysergic Acid Diethylamide