Identification of 5‐Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex

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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 l‐[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 μM [3H]l‐[2‐(4‐azidophenyl)ethyl]‐4‐(3‐trifluoromethyI‐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 prein‐cubated 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.