Large scale expression and purification of the mouse 5-HT3 receptor

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8 mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution. (C) 2013 Elsevier B.V. All rights reserved.

Functional characterization of a human odorant receptor

Valérie Jacquier

Olfaction appears to be mediated by a large family of odorant receptors (ORs) that accounts for ~ 1% of the human genome [1–4]. The functional characterization of these receptors has so far been hindered by their poor functional expression in the plasma membrane of heterologous cells. In order to investigate the problem of inefficient OR membrane targeting, sequential stages in the life cycle of the human OR17-40 were monitored. The modification of the receptor by different tags enabled the direct visualization of its biogenesis and trafficking in HEK293 cells. A multi-color labelling approach, which involved fluorescent subcellular markers as well as the spectral separation of membrane-inserted and intracellularly located receptors, indicated that high overexpression led to the accumulation of the receptor in the ER, whereas low expression levels improved its membrane targeting. To increase the efficiency of OR-mediated signalling, fluorescence-activated cell sorting of a functional OR17-40 – GFP fusion protein was used to separate low from high expressing cells, thus increasing the fraction of odorant-responsive cells up to 80% of the total cell population. Selectively labelled cell surface receptors accumulated over time in intracellular compartments, indicating constitutive receptor internalization. This process, which was independent of receptor activation, occurred along the clathrin-mediated pathway. Moreover, the imaging of single receptors in the plasma membrane indicated that, in absence of ligand, part of the receptors are already confined within small domains of 195 ± 10 nm. This fraction, which increased upon agonist or antagonist binding, probably corresponds to receptors located in clathrin-coated prepits. Odorant molecules structurally related to the cognate agonist helional were tested for their ability to activate OR17-40. It was found that an aldehyde group connected to an aromatic ring via a carbon chain of defined length and containing a methyl group in alpha or beta position is necessary for activating the receptor. In addition, first evidence for an OR17-40-specific antagonism was provided. Finally, OR17-40 activation was monitored in cell-derived native vesicles, opening new ways for assay miniaturization and the development of odorant screenings in a micro-array format.

EPFL2005

Characterization of the 5HT3 receptor in live cells and in vitro

Veronica Ponce de Leon

The serotonin type 3 receptor (5HT3R) is a homopentameric, cation selective channel that mediates fast excitatory transmission in the nervous system. It is a member of the Cys-loop family of receptors that include the nicotinic acetylcholine receptor (nAChR), the γ-amino butyric acid (GABA) type A and C and glycine receptors. The 5HT3 receptor is involved in behavioral and digestive disorders and it is therefore an important therapeutic target. The 5-HT3 receptor was expressed in mammalian HEK cells (chapter 2) stably and transiently, and its expression was optimized to obtain 1.6 x 106 receptors per cell for its solubilisation (chapter 3) and purification (chapter 4). Solubilisation of the receptor was evaluated under different conditions such as using CHAPS and C12E9 detergents and homogenization procedures such as vortexing and thurraxing the membranes. Radioligand binding assays indicated that the amount of solubilised receptor was similar in both cases. The stability of the receptor at different temperatures was evaluated. The receptor was more stable when kept at -20 °C compared to 4 or 37 °C. Purification of the 5-HT3 receptor from HEK cells was performed by tandem IMAC (Immobilized Metal ion Affinity chromatography)/IMAC purification and IMAC/streptactin flow column chromatography. The 5-HT3R was obtained 50% pure and a yield of 10 pmoles of receptor binding sites per gram of starting material (dry cells) was obtained. Purified receptor preparations were homogenous. Finally, the receptor was concentrated 5.5 times, which allowed the incorporation of the receptor in lipid vesicles for further studies. Detergent solubilised 5-HT3R was incorporated in lipid vesicles and the functionality of the receptor was assessed by studying calcium ion permeability of the channel using calcium ion sensitive fluorophores (chapter 5). The effect of the cAMP dependent kinase (PKA), on the 5-HT3R was investigated by measuring the KD of the radioactive ligand [3H]GR65630 to the receptor in cells where PKA was activated or inhibited (chapter 6). Finally a dose-response curve of the binding activity of the 5-HT3R upon activation of the PKA was assayed in both HEK and CHO cells (chapter 6). Radioligand binding assays indicated that upon activation of the PKA, there was a decrease of the KD of 30% indicating an increase in the receptor binding affinity to 5-HT upon activation of the PKA (chapter 6). A kinase and cytoskeleton binding protein PDLI1 that was co-purified with the receptor was found phosphorylated upon PK activation (chapter 6). As PDLI1-like proteins are adaptor proteins between kinases, cytoskeleton proteins and sometimes receptors, the latter may be involved in the kinase activation of the receptor. These results contribute to the optimization of the receptor expression, solubilisation and purification in mammalian cells for its incorporation in lipid vesicles and for further structural studies. In addition, this work gave new insights to the mechanisms of channel regulation by PKA phosphorylation at the cell membrane and the intermediate proteins that are involved.

EPFL2009

Characterization of the 5HT3 receptor in live cells and in vitro

Veronica Ponce de Leon

EPFL2009

Functional characterization of a human odorant receptor

Valérie Jacquier

EPFL2005