Pop, Nathalie and Igel, Patrick and Brennauer, Albert and Cabrele, Chiara and Bernhardt, Guenther and Seifert, Roland and Buschauer, Armin (2011) Functional reconstitution of human neuropeptide Y (NPY) Y-2 and Y-4 receptors in Sf9 insect cells. JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 31 (4). pp. 271-285. ISSN 1079-9893, 1532-4281
Full text not available from this repository. (Request a copy)Abstract
The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R 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 G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y-2) and hPP (Y-4), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R ++ G alpha alpha(i2) and hY(4)R ++ G alpha alpha(i2)/G alpha alpha(o) ++ RGS4, combined with G beta beta(1)gamma gamma(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY13--36) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na<SU++</SU, 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 Y-2 and Y-4 receptor ligands.</.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-COUPLED RECEPTORS; FORMYL PEPTIDE RECEPTOR; PANCREATIC-POLYPEPTIDE RECEPTORS; HIGH CONSTITUTIVE ACTIVITY; HIGH-AFFINITY; PHARMACOLOGICAL CHARACTERIZATION; N-GLYCOSYLATION; FUSION PROTEINS; FOOD-INTAKE; GUINEA-PIG; Steady-state GTPase assay; Gi/G(o)-proteins; RGS protein; Sf9 insect cells; neuropeptide YY2 receptor; neuropeptide YY4 receptor |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy Chemistry and Pharmacy > Institute of Pharmacy > Alumni or Retired Professors > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 05 Jun 2020 04:44 |
| Last Modified: | 05 Jun 2020 04:44 |
| URI: | https://pred.uni-regensburg.de/id/eprint/20504 |
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