Identification of 5-Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex |
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Authors: | Yutaka Takeuchi Wu Yang Jean C Shih |
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Institution: | Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033. |
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Abstract: | 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. |
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Keywords: | Serotonin receptors 5-HT1A receptor PAPP 1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine Azido-PAPP photolabeling Affinity column chromatography |
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