Pharmacological tools for the NPY receptors: [³⁵S]GTPγS binding assays, luciferase gene reporter assays and labeled peptides

The neuropeptide Y (NPY) family comprises the 36-amino acid peptides neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP). In humans, the biological effects of these peptides are mediated by four functionally expressed receptor subtypes, designated Y1, Y2, Y4 and Y5 receptors (Y1R, Y2R, Y4R, Y5R). Among the NPY receptors the Y4R is unique, as it prefers PP over NPY and PYY as the endogenous ligand. PP is predominantly expressed in an endocrine cell type (PP cells) of the pancreas, and is, for example, considered to be involved in satiety signaling, the regulation of food intake and energy metabolism. With this in view, Y4R agonists are discussed as potential anti-obesity drugs. For the characterization of Y2 and Y4R ligands, pharmacological tools are indispensable. Therefore, the first part of this work was aiming at establishing a [35S]GTPγS binding assay and a luciferase gene reporter assay for the hY2R and hY4R, since these assays complement each other by providing different readouts. The second part of this thesis aimed at the development of full length radio- and fluorescence-labeled hPP analogs, which are more stable compared to the endogenous ligand and [Lys4]hPP. In the [35S]GTPγS binding assay at the hY2R, pharmacological data of selected agonists and antagonists were in good agreement with functional studies in the steady-state GTPase assay and the calcium mobilization assay at the hY2R. Most strikingly, the [35S]GTPγS assay at the hY4R revealed potencies of hPP and GW1229, which were lower by a factor 19 and 180, respectively, compared to reported data for the steady-state GTPase assay. Further investigations at the Y4R revealed that the signal-to-noise ratio was not improved by the variation of the Mg2+ ion concentration. Increasing Na+ concentrations led to minor changes of the potency of hPP, whereas [Lys4]hPP and GW1229 were markedly less potent in the presence of sodium. This is in agreement with the data from radioligand binding assays at the hY4R. Nevertheless, the [35S]GTPγS assay is compromised by a low signal-to-noise ratio impairing the robustness of the data. By using a CRE-controlled luciferase reporter gene assay, agonistic and antagonistic activities of several ligands could be determined in HEK293T cells, stably expressing the human Y2 and Y4 receptor, respectively. The conditions were optimized regarding concentration of the vehicle (DMSO or DMF), the forskolin concentration, the period of incubation and the addition of bacitracin as a protease inhibitor. Although a tremendous reduction of the bioluminescene signal by DMSO and DMF became obvious, at concentrations