Immunolabeling of central serotonin 5-HT1D beta receptors in the rat, mouse, and guinea pig with a specific anti-peptide antiserum

• Published in: Journal of neurochemistry Vol. 65; no. 6; p. 2671
• Main Authors: Langlois, X, Gérard, C, Darmon, M, Chauveau, J, Hamon, M, el Mestikawy, S
• Format: Journal Article
• Language: English
• Published: England 01.12.1995
• Subjects: Amino Acid Sequence; Animals; Autoradiography; Binding Sites; Corpus Striatum - metabolism; Dipeptides - metabolism; Guinea Pigs; Immune Sera; Immunologic Techniques; Iodine Radioisotopes; LLC-PK1 Cells; Male; Mice; Molecular Sequence Data; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Serotonin - genetics; Receptors, Serotonin - metabolism; Serotonin - analogs & derivatives; Serotonin - metabolism; Swine; Transfection
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.1995.65062671.x

A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT1B/1D beta receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT1B/1D beta receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin-O-carboxymethylglycyl[125I]iodotyrosinamide ([125I]GTI; a selective 5-HT1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT1B receptors. The remaining 40% of [125I]GTI binding sites were shown to be 5-HT1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT1B/1D beta, but not the 5-HT1D/1D alpha, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT1D beta receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [125I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT1D beta component of [125I]GTI specific receptor binding sites.