Fig. 1. Predicted amino acid sequence of AS1 aligned with the sequences of 5-HT receptors from a number of invertebrate and mammalian sources. Dots indicate identity with AS1. Putative transmembrane domains are overlined and numbered and sequences used for the design of degenerate primers are boxed. Since the length of both the NH₂- and COOH-termini of AS1 are significantly different than most other 5-HT receptors, these regions have been omitted from the alignment for the sake of clarity. In addition, amino acids in the hypervariable third intracellular loop also have been deleted and the number noted in parentheses. Full length AS predicted amino acid sequence is presented in Fig. 3.

Fig. 2. Evolutionary relationships between the predicted amino acid sequence of AS1 and other 5-HT receptors. AS1 was compared with the human 5-HT₁ [42] and 5-HT₂ [43], Drosophila 5-HT₁ [12] and 5-HT₂ [12], Lymnaea 5-HT [13], and 5-HT₂ [14], C. elegans 5-HT₁ [15] and putative 5-HT₂ receptors [44]. Predicted amino acid sequences were aligned excluding the NH₂-termini, the third cytoplasmic loop and COOH-termini using the program DISTANCES and GROWTREE from University of Wisconsin Genetics Computer Group (GCG). The putative A. suum serotonin receptor (AS1) is boxed.

Fig. 3. (A) Membrane-spanning model of AS1 showing amino acids conserved in regions potentially involved in G-protein coupling, receptor desensitization, internalization, palmitoylation and predicted PKA and PKC phosphorylation sites. Question marks (???) indicate the beginning of the different COOH-terminal sequences outlined in B. (B) The predicted amino acid sequences of the different COOH-termini of the individual AS isoforms (AS1, AS2, and AS3).

Fig. 4. Saturation binding of [¹²⁵I]LSD to membranes isolated from COS-7 cells expressing AS1. Saturation binding assays were conducted in 96-well microplates with increasing concentrations of [¹²⁵I]LSD (0.01–3 nM). Total binding and non-specific binding were determined in the absence and presence of 1000-fold excess of cold LSD, respectively. The binding data (K_d and B_max) were derived from three separate experiments each conducted in triplicate and analyzed by curve fitting (Delta Graph, DeltaPoint Inc., 1993).

Fig. 5. Genomic organization of 5htn. Schematic representation of the 5htn gene and the proposed alternative-splicing to yield the different AS isoforms.

Fig. 6. Tissue-specific expression of the putative AS receptor. Poly A⁺ RNA (8 μg) was isolated from tissues dissected from adult A. suum, separated on a 1% formaldehyde agarose gel, transferred to a nylon membrane and hybridized with a [³²P] labelled cDNA coding for AS1. As a control, the filter was rehybridized with a cloned A. suum α-tubulin probe. P, pharynx; M, body wall muscle; O, ovaries plus oviducts; T, testis; I, intestine.

Fig. 7. Tissue-specific expression of the individual AS isoforms. RT-PCR was conducted as described in Section 2 using cDNA pools prepared from poly A⁺ RNA isolated from dissected pharynx and body wall muscle. The identities of the individual bands were confirmed by direct sequencing. Upper panel, primers including the region coded for by exon IV; lower panel, primers specific for the carboxy termini of AS2/3. P, pharynx; M, body wall muscle.

Table 1. Tissue-specificity of alternatively-spliced full length AS isoformsa