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

JR Raymond, A Fargin, MJ Lohse, JW Regan, SE Senogles, RJ Lefkowitz and MG Caron

Howard Hughes Medical Institutes Laboratories, Duke University Medical Center, Durham, North Carolina 27710.

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.

Volume 36, Issue 1, pp. 15-21, 07/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				J. H. Turner, M. N. Garnovskaya, S. D. Coaxum, T. M. Vlasova, M. Yakutovich, D. M. Lefler, and J. R. Raymond Ca2+-Calmodulin and Janus Kinase 2 Are Required for Activation of Sodium-Proton Exchange by the Gi-Coupled 5-Hydroxytryptamine1a Receptor J. Pharmacol. Exp. Ther., January 1, 2007; 320(1): 314 - 322. [Abstract] [Full Text] [PDF]

				A. J. Barr and D. R. Manning Agonist-independent Activation of Gz by the 5-Hydroxytryptamine1A Receptor Co-expressed in Spodoptera frugiperda Cells. DISTINGUISHING INVERSE AGONISTS FROM NEUTRAL ANTAGONISTS J. Biol. Chem., December 26, 1997; 272(52): 32979 - 32987. [Abstract] [Full Text] [PDF]