ABSTRACT
The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.
Subject(s)
Chromosomes, Human, Pair 3/genetics , Multigene Family/genetics , Receptors, Serotonin, 5-HT3/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Caco-2 Cells , Cell Line, Tumor , Cloning, Molecular , Exons , Female , Gene Expression , Genes/genetics , Humans , Introns , Male , Molecular Sequence Data , Phylogeny , Radiation Hybrid Mapping , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Presenilin (PS1 or PS2) is an essential component of the active gamma-secretase complex that liberates the Abeta peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the gamma-secretase activity in the presence or absence of gamma-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Abeta42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the gamma-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Abeta38 and Abeta42, but to reduce levels of Abeta39, showing a pattern of Abeta formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the gamma-site without interfering with Notch processing.