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 dr amatically stimulated by serotonin (5-HT). In the present study, a cDNA poo l was prepared from poly A(+) RNA isolated from pharynxes dissected from th e parasitic nematode, Ascaris suum, and was used as a template for RT-PCR w ith degenerate primers designed from sequences conserved in 5-HT receptors from a Variety of sources. A putative 5-HT receptor cDNA (ASI) was identifi ed 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' unt ranslated region of 127 nucleotides with a polyadenylation signal (ATTAAA) and a short poly A(+) tail. The coding region predicted a protein of 532 am ino acids with a molecular weight of 60 176. When ASI was transiently expre ssed in COS-7 cells, isolated membranes exhibited the high affinity, satura ble binding of [I-125]LSD. More importantly, [I-125]LSD binding was inhibit ed by 5-HT, but not other biogenic amines, supporting the identification of AS1 as a 5-HT receptor. Additional cDNAs identical, in part, to ASI were a lso identified. AS1 Delta IV lacked a predicted 42 amino acids at the carbo xy terminus of the third intracellular loop while AS2 and AS3 contained dif ferent COOH-termini, regions implicated in G-protein coupling in other hept ahelical receptors. A portion of the gene (5htn) encoding ASI also was clon ed and sequenced. This genomic fragment was about 10 kb, contained the enti re AS1 open reading frame and included eight exons and seven introns. From this analysis, it appears that these different AS cDNAs were generated by a lternative-splicing, AS1 Delta IV from the deletion of exon IV, and AS2 and AS3 from the use of alternative sites within exon VII as 5' splice accepto r sites for exon VIII. Using RT-PCR and primers specific for each of the is oforms, AS1-3 appeared to be expressed in pharynx, while only AS1 and AS2 w ere 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 . (C) 1999 Elsevier Science B.V. All rights reserved.