title: Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors.
abstract: We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine3 (5-HT3) receptor subunit, thus permitting, for the first time, localization of 5-HT3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT3 receptors, whilst Western blots of recombinant 5-HT3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT3 receptors from N1E-115 cell membranes produced a broad band at 50-54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT3 receptors. Studies on native 5-HT3 receptors revealed that pAb120 could recognize 5-HT3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT3 receptor neurobiology.
authors.affiliation: Division of Neurobiology, Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, CB2 2QH, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Spier
authors.forename: G
authors.initials: G
authors.lastname: Wotherspoon
authors.forename: S V
authors.initials: SV
authors.lastname: Nayak
authors.forename: R A
authors.initials: RA
authors.lastname: Nichols
authors.forename: J V
authors.initials: JV
authors.lastname: Priestley
authors.forename: S C
authors.initials: SC
authors.lastname: Lummis
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008297:Male; D017729:Presynaptic Terminals / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology
chemical_list: D008024:Ligands; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins