Identification of 5-hydroxytryptamine1A receptor proteins in bovine frontal cortex.
5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.
Takeuchi
Y
Y
Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033.
Yang
W
W
Shih
J C
JC
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005625:Frontal Lobe / Q000032:analysis*; D008970:Molecular Weight; D010777:Photochemistry; D010879:Piperazines; D011985:Receptors, Serotonin / Q000032:analysis* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D012995:Solubility
D000345:Affinity Labels; D001386:Azides; D010879:Piperazines; D011985:Receptors, Serotonin; C050004:1-(2-(4-azidophenyl)ethyl)-4-(3-trifluoromethylphenyl)piperazine; D012701:Serotonin; C044643:LY 165163