Development and Characterization of Monoclonal Antibodies Specific to the Serotonin 5-HT_2AReceptor

Abstract

SUMMARY Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT 2A receptor. To generate MAbs against 5-HT 2A R, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT 2A R recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT 2A R, rat and human brain lysate, and lysate from cell lines transfected with 5-HT 2A R cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT 2A R N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT 2A R in lysates from rat brain and a 5-HT 2A R-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT 2A R but not with recombinant 5-HT 2B R and 5-HT 2C R. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies. (J Histochem Cytochem 46:811-824, 1998)

Characterization of Serotonin Receptors in Pregnant Human Myometrium

“…Radioligand binding demonstrated specific binding of [ 3 H]ketanserin, and this was estimated as approximately 34 fmol/mg protein. Antibodies specific for the 5-HT 2A receptor detected a protein doublet at around 55 kDa, which was consistent with the predicted molecular mass of the 5-HT 2A receptor (53 kDa) and with previous reports (Wu et al, 1998). Immunohistochemistry localized this receptor to the smooth muscle cells of the myometrium, although not all muscle cells were labeled.…”

Identification of Amino Acid Residues Important for Sarpogrelate Binding to the Human 5-Hydroxytryptamine2A Serotonin Receptor

“…Western blots probed with the anti-5-HT 2A R antibody detected an immunoreactive band of about 55 kDa in both the wild-type and mutant receptor-expressing cells (Fig. 5: A and B), as reported earlier for this antibody in NIH-3T3 cells (15), suggesting that the mutations in TMH 3 did not cause any abnormality in the receptor proteins. It is also suggested that the mutant proteins are inactive but are still expressed and detectable by antibody.…”

Neuronal localization of 5-HT type 2A receptor immunoreactivity in the rat basolateral amygdala

“…The monoclonal antibody to 5-HT 2A R (clone G186-117; BD Pharmingen # 556326) was raised against a recombinant fusion protein of glutathione S-transferase and a peptide containing amino acids 1-76 of the human 5-HT 2A R receptor (Wu et al, 1998), although a recent report indicates that the targeted epitope is comprised of a sequence between amino acids 64-71 (Cornea-Hébert et al, 2002). The specificity of the antibody was determined in Western blot analyses; it labeled a single band of 55 kDa.…”

“…The specificity of the antibody was determined in Western blot analyses; it labeled a single band of 55 kDa. Staining was completely eliminated by preadsorption of the antibody with the GST-5HT 2A R fusion protein (200 g protein/100g antibody), but not with GST-5HT 2B R or GST-5HT 2C R fusion proteins (Wu et al, 1998).…”