Immunolabeling of central serotonin 5-HT1D beta receptors in the rat, mouse, and guinea pig with a specific anti-peptide antiserum.
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.
Langlois
X
X
INSERM U. 288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Gérard
C
C
Darmon
M
M
Chauveau
J
J
Hamon
M
M
el Mestikawy
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D003342:Corpus Striatum / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D006168:Guinea Pigs; D007106:Immune Sera; D007158:Immunologic Techniques; D007457:Iodine Radioisotopes; D018374:LLC-PK1 Cells; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000031:analogs & derivatives / Q000378:metabolism; D013552:Swine; D014162:Transfection
D004151:Dipeptides; D007106:Immune Sera; D007457:Iodine Radioisotopes; D011985:Receptors, Serotonin; C072128:serotonin-O-carboxymethyl-Gly-Tyr; D012701:Serotonin