Localization of the 5-hydroxytryptamine2C receptor protein in human and rat brain using specific antisera.
The mouse 5-HT2C receptor and its third and fourth (C-terminal) cytoplasmic domain have been expressed as fusion proteins in bacteria. After purification antisera were generated against the fusion proteins. Characterization by immunoblotting using eukaryotic cells expressing the 5-HT2C and 5-HT2A receptors showed that high titer antibodies could be obtained only against the third and fourth cytoplasmic domain but not the entire receptor. Affinity purified antibodies were used to study the location of 5-HT2C receptors in rat and human brain sections. This distribution was compared with the location of 5-HT2C receptor binding sites as determined by [3H]mesulergine, a 5-HT2C receptor radioligand. The antibodies recognized sites in the rat choroid plexus, hippocampus, cerebral cortex, striatum and substantia nigra with a similar distribution as the 5-HT2C binding sites. One antiserum directed against the 5-HT2C receptor C-terminus crossreacted with the human receptor protein in immunoblots. In human brain sections it labelled sites including cerebral cortex, substantia nigra and cerebellum. Our results demonstrate that the antibodies are suitable to identify 5-TH2C receptors in rat and human brain. They visualize a protein distribution which correlates well with the location of the 5-HT2C receptor binding sites as would be expected if affinity states do not influence the binding pattern.
Abramowski
D
D
Sandoz Pharma Ltd, Preclinical Research, Basle, Switzerland.
Rigo
M
M
Duc
D
D
Hoyer
D
D
Staufenbiel
M
M
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D002478:Cells, Cultured; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis*; D007150:Immunohistochemistry; D008297:Male; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011985:Receptors, Serotonin / Q000032:analysis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014162:Transfection
D007106:Immune Sera; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins