Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-( sup 125 I) iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

• Published in: Molecular pharmacology Vol. 36:1
• Main Authors: Raymond, J.R., Fargin, A., Lohse, M.J., Regan, J.W., Senogles, S.E., Lefkowitz, R.J., Caron, M.G.
• Format: Journal Article
• Language: English
• Published: United States 01.07.1989
• Subjects: 550201 - Biochemistry- Tracer Techniques; AFFINITY; AMINES; ANIMALS; AROMATICS; AUTONOMIC NERVOUS SYSTEM AGENTS; AZAARENES; AZIDES; AZOLES; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BIOASSAY; CHEMICAL COMPOSITION; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; IMMUNOASSAY; INDOLES; INTERMEDIATE MASS NUCLEI; IODINE 125; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LIGANDS; MAMMALS; MAN; MEMBRANE PROTEINS; NEUROREGULATORS; NITROGEN COMPOUNDS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PRIMATES; PROTEINS; PYRROLES; RADIOISOTOPES; RADIOPROTECTIVE SUBSTANCES; RECEPTORS; SEROTONIN; SYMPATHOMIMETICS; TRACER TECHNIQUES; TRYPTAMINES; VERTEBRATES
• ISSN: 0026-895X; 1521-0111

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-(125I)iodophenethyl)spiperone (( 125I)N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by (125I)N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block (125I)N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand (125I)N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.