Abstract
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-125Iiodophenethyl)spiperone
( 125IN3-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 125IN3-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 125IN3-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
125IN3-NAPS and of a potent immunoprecipitating antiserum (JWR21)
should greatly facilitate the biochemical characterization of the
human 5-HT1A receptor.
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