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.
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.
Raymond
J R
JR
Howard Hughes Medical Institutes Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
Fargin
A
A
Lohse
M J
MJ
Regan
J W
JW
Senogles
S E
SE
Lefkowitz
R J
RJ
Caron
M G
MG
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007457:Iodine Radioisotopes; D011233:Precipitin Tests; D011954:Receptors, Dopamine / Q000032:analysis; D017448:Receptors, Dopamine D2; D011985:Receptors, Serotonin / Q000032:analysis* / Q000276:immunology; D013134:Spiperone / Q000031:analogs & derivatives / Q000378:metabolism*; D013764:Tetrahydronaphthalenes / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D011985:Receptors, Serotonin; D013764:Tetrahydronaphthalenes; D013134:Spiperone; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C044567:N-(4-azido-3-iodophenethyl)spiperone