Functional reconstitution of human neuropeptide Y (NPY) Y₂and Y₄receptors in Sf9 insect cells

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?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?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(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.

Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists

“…27 was obtained as a yellowish oil (R f = 0.37 in DCM) (131 mg, 44%).C 29 H 51 N 13 O 2 S 2 Â 5 TFA (1248.03).4.2.14. General procedure for the synthesis of the bivalent fluorescence ligands(29)(30)(31) The reactions were carried out in a 1.5-mL Eppendorf reaction vessel. The amine precursor 28 (2 equiv) was dissolved in 30 lL DMF and NEt 3 (15 equiv) was added.…”

[3H]UR‐DE257: Development of a Tritium‐Labeled Squaramide‐Type Selective Histamine H2 Receptor Antagonist

“…[52,53]) expressing the hH 2 R-G saS or the gpH 2 R-G saS and on the isolated spontaneously beating guinea pig right atrium [46,54] were performed as previously described, except that the incubation period of the guinea pig atrium in the presence of the antagonists was extended to 60 min. Binding data on recombinant histamine receptor subtypes and orthologues expressed in Sf9 cells (hH 1 R + RGS4; hH 2 R-G saS , gpH 2 R-G saS , rH 2 R-G saS ; hH 3 R + G ia2 + b 1 g 2 ; hH 4 R + G ia2 + b 1 g 2 ) or HEK293T CRE-Luc cells [55] (hH 2 R) using the following radioligands: H 1 R,[ …”

Abolishing Dopamine D2long/D3 Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2 Receptor Agonists

“…Competition binding experiments were performed with membrane preparations of Sf9 insect cells, expressing the h H 1 R + RGS4, h H 2 R-G sαS fusion protein, h H 3 R + G αi2 + G β1γ2 , or the h H 4 R + G αi2 + G β1γ2 . General procedures for the generation of recombinant baculoviruses, the culture of Sf9 cells, and membrane preparations have been described elsewhere . The competition binding experiments were performed as previously described in detail , with one minor modification: PBS (8 g NaCl, 0.2 g KCl, 1.0 g Na 2 HPO 4 ·2H 2 O, 0.15 g NaH 2 PO 4 ·H 2 O, 0.1 g KH 2 PO 4 in 1 L Millipore H 2 O; pH 7.4; 4 °C) was used as washing buffer while harvesting instead of the previously used binding buffer. , [ 3 H]­Mepyramine (specific activity (spec.…”

“…1 H NMR (600 MHz, DMSO-d 6 ) δ 10.26 (br s, 2H), 9.02 (br s, 2H), 8.50 (br s, 4H), 7.97 (br s, 3H), 7.49 (br s, 2H), 3.29 (q, J = 6.7 Hz, 4H), 3.07 (q, J = 6.6 Hz, 4H), 2.85 (t, J = 7.5 Hz, 4H), 1. 88 General Procedure for the Synthesis of Oxadiazole Derivatives 37 and 58. The oxadiazole heterocycle was formed according to a previously published procedure.…”