title: Identification of a novel 5-HT(4) receptor splice variant (r5-HT(4c1)) and preliminary characterisation of specific 5-HT(4a) and 5-HT(4b) receptor antibodies.
abstract: The human 5-hydroxytryptamine (5-HT(4)) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (+/-)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.
authors.affiliation: Neuroscience Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. alison.2.reid@gsk.com
authors.forename: Alison M
authors.initials: AM
authors.lastname: Ray
authors.forename: Rosemary E
authors.initials: RE
authors.lastname: Kelsell
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Houp
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Kelly
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Medhurst
authors.forename: Helen M
authors.initials: HM
authors.lastname: Cox
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Calver
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D041981:Gastrointestinal Tract / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D044422:Receptors, Serotonin, 5-HT4 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D012333:RNA, Messenger; D017366:Serotonin Receptor Agonists; D044422:Receptors, Serotonin, 5-HT4; D000242:Cyclic AMP