It is now well accepted that at least some serotonin receptors exist in dimeric and oligmeric forms. The linking of receptor ligands has been shown to have potential in the development of selective agonists and antagonists for traditionally refractive receptors. Here we report the development of a dimeric version of the known 5-HT2AR antagonist, M-100907. Derivatives of M-100907 were synthesized to determine an appropriate site for the linker connection. Then, homodimers with polyether linkers of different lengths were functionally tested in a bioassay to determine the optimal linker length. Attachment at the catechol of M-100907 with linkers between 12 and 18 atoms in length proved to be optimal.

J. Neurochem. (2010) 113, 1504–1515. Abstract The action of serotonin (5‐HT) at the 5‐HT2C receptor (5‐HT2CR) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5‐HT2CR protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5‐HT2CR in synaptosomes and the post‐synaptic density, an electron‐dense thickening specialized for post‐synaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, post‐synaptic density), and controls (peptide neutralization, 5‐HT2CR stably‐expressing cells), we established the selectivity of two commercially available 5‐HT2CR antibodies and employed the antibodies in western blot and immunoprecipitation studies of prefrontal cortex (PFC) and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5‐HT2CR in post‐synaptic density‐enriched fractions from both PFC and motor cortex. Co‐immunoprecipitation studies revealed the presence of post‐synaptic density‐95 within the 5‐HT2CR protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5‐HT2CR is localized within the post‐synaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.

BackgroundThe serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.ResultsWe optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.ConclusionsThese data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.