Development of a Specific 5‐HT7 Receptor Antibody: Is This Even Possible?

Abstract

Our laboratory has a vested interest in measuring the location and expression of the 5‐hydroxytryptamine (5‐HT, serotonin) 7 (5‐HT7) receptor. The 5‐HT7 receptor is responsible for the fall in blood pressure when 5‐HT is infused acutely or chronically in the rat. Determining tissue‐specific receptor expression would aid in understanding mechanisms that support the 5‐HT7 receptor‐mediated fall in blood pressure. We have, using tissues from a 5‐HT7 receptor KO rat we created, tested over a dozen commercially available 5‐HT7 receptor antibodies. None can discriminate between WT and KO tissues, indicating a lack of specificity that has been found commonly with antibodies directed against G protein coupled receptors (GPCRs), of which the 5‐HT7 receptor is one. We contracted with 7TM antibodies (Germany) to develop rigorously a 5‐HT7 receptor specific antibody, knowing there was a 50% chance of failure. Three different antigens, two targeting the third internal loop and one the C terminus, were used in rabbits to generate antibodies. We sent 7TM a plasmid for the r5‐HT7 receptor which, transiently expressed in HEK293 cells, served as a positive control for screening initial antibodies. In Western analyses done in Germany, nine (9) antibodies (all antigens represented) detected a ~75 kDa protein that was not present in homogenates of non‐transfected HEK293 cells. These same antibodies were sent to MSU for testing in the following ways: analyses of r5‐HT7 receptor expressing HEK293 cells to demonstrate protein dependence of antibody binding in Westerns; immunohistochemical analyses of brains from 5‐HT7 receptor WT and KO rats. Only antibodies that recognized the C terminus of the 5‐HT₇ receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT] positively and concentration‐dependently identified the r5‐HT7 receptor expressing HEK293 cells in Westerns. By contrast, these same C‐terminus antibodies have not specifically detected the 5‐HT7receptor in rat tissues. No specificity of signal was observed between brain sections of WT and KO rats when tested immunohistochemically. Future studies will explore different protein isolation and immunoprecipitation in buffers specialized for GPCR extraction with the goal of increased specificity of antibody signal.