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Abstract
The four functionally expressed human neuropeptide Y receptor subtypes (hY1R, hY2R, hY4R, hY5R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive Gi/o-proteins. The number of small molecules described as ligands for hY1R and hY5R exceeds by far those for hY2R. Potent non-peptidergic ligands for the hY4R are not available so far. Here, we report on the functional reconstitution of the hY2R and the hY4R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the Gi/o family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y2) and hPP (Y4), the GPCRs coupled to various Gi/Go-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY2R + Gαi2 and hY4R + Gαi2/Gαo + RGS4, combined with Gβ1γ2, yielded best signal-to-noise ratios. hY2R function was validated using both agonistic peptides (NPY, PYY, NPY13–36) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY4R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY2R and abolished hY4R function. Investigations with monovalent salts showed sensitivity of hY4R toward Na+, revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y4R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y2 and Y4 receptor ligands.
Acknowledgements
The authors thank Gertraud Wilberg, Nina Kraupner, and Stefan Weidinger for technical assistance and Dr. Erich Schneider and Dr. David Schnell for stimulating discussions. This work was supported by the Graduate Training Program (Graduiertenkolleg) GRK760, “Medicinal Chemistry: Molecular Recognition—Ligand–Receptor Interactions” of the Deutsche Forschungsgemeinschaft (DFG).
Declaration of interest
The authors declare no conflict of interest.