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

J. R. Raymond, A. Fargin, M. J. Lohse, John W Regan, S. E. Senogles, R. J. Lefkowitz, M. G. Caron

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15-21
Number of pages7
JournalMolecular Pharmacology
Volume36
Issue number1
StatePublished - 1989
Externally publishedYes

Fingerprint

Spiperone
Receptor, Serotonin, 5-HT1A
Ligands
COS Cells
Immune Sera
Frontal Lobe
Immunoprecipitation
Hippocampus
Dopamine D2 Receptors
Population Groups
Amino Acid Sequence
Cell Membrane
Pharmacology
Rabbits
Peptides
azido-N-(4-aminophenethyl)spiperone
Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

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. / Raymond, J. R.; Fargin, A.; Lohse, M. J.; Regan, John W; Senogles, S. E.; Lefkowitz, R. J.; Caron, M. G.

In: Molecular Pharmacology, Vol. 36, No. 1, 1989, p. 15-21.

Research output: Contribution to journalArticle

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abstract = "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.",
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