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

Journal Article · Sat Jul 01 00:00:00 EDT 1989 · Molecular Pharmacology; (USA)

OSTI ID:5641637

Raymond, J R; Fargin, A; Lohse, M J; Regan, J W; Senogles, S E; Lefkowitz, R J; Caron, M G ^[1]

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1. Duke Univ. Medical Center, Durham, NC (USA)

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.

• OSTI ID: 5641637
• Journal Information: Molecular Pharmacology; (USA), Vol. 36:1; ISSN 0026-895X
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RECEPTORS
CHEMICAL COMPOSITION
TRYPTAMINES
AFFINITY
AZIDES
IMMUNOASSAY
IODINE 125
LIGANDS
MAN
SEROTONIN
TRACER TECHNIQUES
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
AZOLES
BETA DECAY RADIOISOTOPES
BIOASSAY
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INDOLES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MEMBRANE PROTEINS
NEUROREGULATORS
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PRIMATES
PROTEINS
PYRROLES
RADIOISOTOPES
RADIOPROTECTIVE SUBSTANCES
SYMPATHOMIMETICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques