Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe. Mol Pharmacol 1989 Jul;36(1):15-21
Date
07/01/1989Pubmed ID
2526292Scopus ID
2-s2.0-0024362284 (requires institutional sign-in at Scopus site) 27 CitationsAbstract
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.
Author List
Raymond JR, Fargin A, Lohse MJ, Regan JW, Senogles SE, Lefkowitz RJ, Caron MGAuthor
John R. Raymond MD President, CEO, Professor in the President department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
8-Hydroxy-2-(di-n-propylamino)tetralinAffinity Labels
Azides
Cell Line
Humans
Iodine Radioisotopes
Precipitin Tests
Receptors, Dopamine
Receptors, Dopamine D2
Receptors, Serotonin
Spiperone
Tetrahydronaphthalenes