Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine 1A receptor

Abstract

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine 1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine 1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [ 3H]8-hydroxy-2-(di- n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine 1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine 1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine 1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine 1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine 1A receptors might differ from one neuronal cell type to another.