The human histamine H₂‐receptor couples more efficiently to Sf9 insect cell G_s‐proteins than to insect cell G_q‐proteins: limitations of Sf9 cells for the analysis of receptor/G_q‐protein coupling

Abstract

The human histamine H 2 -receptor (hH 2 R) couples to G s -proteins to activate adenylyl cyclase and to G q -proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH 2 R to insect and mammalian G s -and G q -proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH 2 R with mammalian G proteins was assessed with coexpressed proteins or receptor-G a fusion proteins that enhance coupling efficiency. hH 2 R efficiently coupled to insect G s -proteins to activate adenylyl cyclase. However, hH 2 R poorly coupled to insect G q -proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H 1 -receptor (H 1 R) from man and guinea pig. The measurement of intracellular free Ca 2+ concentration was not useful for studying receptor/G q -protein coupling. hH 2 R also efficiently interacted with mammalian G s -proteins, specifically with fused G sa as assessed by guanosine 5¢-O-(3-thiotriphosphate) (GTPcS)-sensitive highaffinity agonist binding, agonist-stimulated [ 35 S]GTPcS binding and adenylyl cyclase activation. In contrast, coupling of hH 2 R to coexpressed and fused mammalian G qa was poor. However, our inability to reconstitute efficient coupling of PAFR and H 1 R to mammalian G qa indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH 2 R couples more efficiently to insect cell G s -proteins than to insect cell G q -proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian G s -and G q -proteins and assessing G q -mediated activation of effector systems. The histamine H 2 -receptor (H 2 R) is a prototypical neurotransmitter receptor that belongs to the superfamily of G protein-coupled receptors (GPCRs) (Leurs et al. 1995; Hill coupled receptor; gpH 1 R, guinea pig histamine H 1 -receptor; gpH 2 R, guinea pig histamine H 2 -receptor; G q -proteins, family of G proteins (comprising mammalian G qa , G 11a , G 14a , G 15a and G 16a and insect G qproteins) that mediates phospholipase C activation; G s -proteins, family of G proteins (comprising mammalian G sa-short , G sa-long , G sa-extralong and G aolf and insect G s -proteins) that mediates adenylyl cyclase activation; G saL , long splice variant of the G s -protein G sa ; G saS , short splice variant of the G s -protein G sa ; GTPcS, guanosine 5¢-O-(3-thiotriphosphate); hH 1 R, human histamine H 1 -receptor; hH 2 R, human histamine H 2 -receptor; hH 2 R-G saL , fusion protein containing the human histamine H 2 -receptor and the long splice variant of G sa ; hH 2 R-G saS , fusion protein containing the human histamine H 2 -receptor and the short splice variant of G sa ; ...

Histamine H4 receptor–RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

“…We previously observed a similar effect, when hH 1 R or gpH 1 R were co-expressed with the regulators of G-protein signaling RGS4 and GAIP in Sf9 cell membranes [27] .…”

Multiple Differences in Agonist and Antagonist Pharmacology between Human and Guinea Pig Histamine H1-Receptor

“…Differences between Human and Guinea Pig H 1 -Receptor 1105 at ASPET Journals on May 12, 2018 jpet.aspetjournals.org (Houston et al, 2002). Dimethindene enantiomers were a kind gift of Dr. G. Lambrecht (Department of Pharmacology, University of Frankfurt/Main, Germany).…”

“…As read-out, we focused on the determination of the GTPase activity of insect cell G qproteins in the presence of the RGS proteins RGS4 and GAIP. This coexpression system provides a sensitive model for studying H 1 R at the G-protein level (Houston et al, 2002). The GTPase assay is a steady-state method and eliminates the impact of effector availability/compartmentation and pharmacokinetic barriers on the properties of agonists (Buschauer, 1989;Ostrom et al, 2000).…”

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…Cell Culture and Membrane Preparation-Sf9 cells were cultured and infected with 1:100 dilutions of high titer virus stocks as described (45). Sf9 membranes were prepared as described (46) and stored at Ϫ80°C until use.…”