Identification of the ligand-binding subunit of the human 5-hydroxytryptamine(1A) receptor with N-(p-Azido-m-[¹²⁵I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

The ligand-binding subunit of the human 5-hydroxytryptamine(1A) (5-HT(1A)) receptor transiently expressed in COS-7 cells and of the native human 5-HT(1A) receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[¹²⁵]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-HT(1A) receptor. In transiently transfected COS-7 cells expressing 14 ± 3 pmol/mg of protein human 5-HT(1A) receptors, a single broad 75-kDa band was photoaffinity labeled by [¹²⁵I]N₃-NAPS. This band displayed the expected pharmacology of the 5-HT(1A) receptor, as evidenced by the ability of a series of competing ligands to block [¹²⁵I]N-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-HT(1A) receptor.