Introduction

Several approaches to the development of binding assays for the Y2 receptor have been reported in the literature. Some authors use tissue preparation, e.g. rat forebrain tissue (Parker et al., 2002b), rabbit kidney membranes (Beck-Sickinger et al., 1992), rat jejunal crypt cells (Goumain et al., 2001) or human frontal cortex membrane homogenate (Dumont et al., 2000). Disadvantages of these preparations are the time consuming preparation procedure and the presence of other receptors especially NPY receptor subtypes, interfering with ligand binding to the Y2 receptor.

To circumvent this problem, Y2 selective labeled ligands are used, and the pharmacological binding profile is determined with known NPY receptor ligands.

Nevertheless, it cannot be excluded that binding is partly mediated by other receptor subtypes than the Y2 receptor. The frequently used Y2 receptor ligand hPYY binds with the same affinity to hY1 and hY2 receptors and even the Y2 preferring ligand hPYY3-36 shows considerable affinity to the Y1 receptor (Ki = 0.24 nM for the hY2

receptor vs. Ki = 13.3 nM for the hY1 receptor) as has been reported by Gehlert and co-workers (Gehlert et al., 1996a).

Another receptor source for binding assays are cells constitutively expressing the Y2

receptor, namely the astrocytoma cell line LN319 (Beck-Sickinger et al., 1992) and the two neuroblastoma cell lines SMS-KAN (Shigeri and Fujimoto, 1994) and CHP234 (Lynch et al., 1994). The receptor expression is sufficient for radioligand binding assays (especially when membrane preparations are used) but the aforementioned cell lines are not suited when higher expression levels of the Y2

receptor are required, e.g. in cellular assays.

3.1.1.1 Heterologous expression systems

Heterologous expression has become an invaluable tool for the establishment of receptor binding assays. Recombinant GPCRs have been expressed in bacteria (Marullo et al., 1988), yeast (Weiss et al., 1995), insect cells (Figler et al., 1996), frog oocytes (Lee and Durieux, 1998) and mammalians cells (Neve and Neve, 1998).

Although all of these expression systems produce receptors, there are substantial differences in expression levels and post-translational modification of the resulting

receptor proteins. For example, bacteria lack the machinery for protein glycosylation, which is a requirement for the correct folding and transport of some receptors to the cell surface (Tifft et al., 1992). Nevertheless, N-glycosylation is not always essential, and several GPCRs have been expressed in E. coli (for an overview, see Grisshammer, 1998). In insect cells proteins are post-translationally glycosylated, but these cells lack the galactose and sialic acid transferase and are therefore unable to convert N-linked oligosaccharides to complex sugars (Miller, 1988), resulting in GPCRs glycosylated to a lesser degree than their mammalian counterparts (Kobilka, 1995). Concerning post-translational modifications, mammalian cells are best suited for the study of GPCRs in binding and functional assays.

3.1.1.2 Transient versus stable transfection

Heterologous expression of a GPCR in mammalian cells can be achieved by stable or transient transfection. In a transient expression system high levels of the gene product are expressed over a limited period of time shortly after transfection (usually 2-4 days). Compared to stable transfection, in most cases this method yields higher expression levels which can be attained much more rapidly since the time consuming procedure to generate, isolate and characterize transfected cells is not required.

Therefore, transient expression is often used for the purification of receptors, for the development of antibodies or as a receptor source for binding or functional assays.

One drawback of transient expression is the fact that the transfection efficiency can vary considerably depending on the purity of the DNA preparations and the state of the cells to be transfected. Furthermore, the ratio of recombinant receptors to other cellular components can not be maintained at a constant level. This variable stoichometrie can interfere especially with functional assays. Stably transfected cells have incorporated the receptor cDNA into the genome and propagate it with each mitotic event. Therefore, a defined, constant quantity of the recombinant receptor is expressed for many generations. Another advantage of stable vs. transient transfection is that once a cell line, stably expressing the receptor, is generated, the laborious transfection procedure is not required anymore.

3.1.1.3 Choice of the host cell

The host cell line into which the GPCR is to be transfected should not endogenously express other receptor subtypes which could interfere with the binding of the ligand.

This allows the determination of binding and functional data on a null background.

Other features of a host cell line to be considered are transfection efficiency, growth rate and routine maintenance. Concerning clonal selection, the cells should be able to grow at a very low density. Suspension cells are easier to culture and more convenient for the use in many applications, but in most cases they are more difficult to transfect. Most of the available cell lines are adherently growing cells, and in most cases standard cell lines as e.g. HEK 293, CHO-K1 or COS-7 are used.

3.1.1.4 Choice of the expression vector

There are many commercially available vectors that will enable expression of receptor cDNA in mammalian cells. Most of them contain a ColE1 or pUC derived origin of replication for propagation and the ampicillin resistance gene, encoding β-lactamase for selection in Escherichia coli. The three key features promoter, transcription termination and RNA processing signals, and a selectable marker should be considered when selecting an appropriate expression vector. Viral enhancer-promoter sequences from the human cytomegalovirus (CMV) immediate early gene, the simian virus (SV-40) early gene, and the Rous sarcoma virus long terminal repeat (RSV LTR) are commonly used to achieve high levels of constitutive expression of the receptor. The cDNA is incorporated into the vector in the sense direction downstream of viral promoter elements and upstream of a polyadenylation signal. Although these promoters work well in a wide variety of cell lines, the suitability of a promoter depends on the host cell line used for transfection and should be tested if necessary. This also holds for RNA processing signals which are necessary for the stability of the transgenic mRNA. Polyadenylation signal and transcription termination signal sequences are commonly taken from SV-40 or the bovine growth hormone (BGH) gene. Dominant selectable markers which confer resistance to certain antibiotics are used to isolate stable transfectants. In most of the modern expression vectors the drug-resistance cassette is included in the same vector containing the cDNA of interest. However, it is also possible to co-transfect an expression vector with another vector containing the drug-resistance cassette.

Commonly used selection markers are resistance to neomycin (G418), zeocin, blastocidin, hygromycin, puromycin and bleomycin.

Usually, only the protein-coding region of the cDNA is inserted into the expression vector. Non-coding sequences upstream the translational start site may contain regulatory regions compromising gene expression. Also the 3’ untranslated region may impair gene expression, but this region is usually less problematic. The insertion of a Kozak’s consensus sequence (Kozak, 2002) at the translational start site can often enhance the expression of a foreign gene.

3.1.1.5 Transfection of mammalian cells

Many different techniques are known by which exogenous DNA can be incorporated and expressed by mammalian cells. These include direct methods like microinjection of RNA and DNA, transfection by calcium phosphate precipitation or lipofection, electroporation and particle-bombardment-mediated gene transfer. The cheapest method is the standard CaPO4 precipitation (Chen and Okayama, 1987), but in most cases higher transfection efficiencies can be achieved by electroporation or lipid-mediated gene transfer reagents. Usually, each transfection protocol has to be optimized for each cell line. An alternative method is the use of viral vectors.

Compared to mechanical and chemical transfection strategies, the use of recombinant viruses is very effective as viruses have evolved successful mechanisms for entering cells, transferring genetic material, and optimizing expression of the exogenous (viral) proteins. On the other hand, the virus-based systems are more challenging in terms of vector construction and the optimization of transfection efficiency.

3.1.1.6 Selection and screening of cell clones

Usually selection of transfected cells is carried out 2-3 days after transfection allowing the transfected cells to express the drug-resistance cassette. As drug sensitivity markedly varies among cell lines, the concentration of the antibiotic sufficient to kill the non-transfected wild type cells should be determined prior to the transfection or non-transfected cells should be incubated with the antibiotic as a control in parallel. Resistant cell clones are selected and propagated, but because

the expression of the recombinant receptor can vary, the cell clones should be screened in binding or functional assays.

Human Y2 receptors have been expressed in HEK293 (Berglund et al., 2002;

Dautzenberg et al., 2005; Dumont et al., 2000), COS-7 and CHO (Gerald et al., 1995;

Goumain et al., 2001; Rose et al., 1995) cells. In each case the authors used membrane preparations for the binding assays except for Rose and co-workers, who used whole cells.