Abstract
Numerous structurally diverse ligands were developed to target the human histamine H3 receptor (hH3R), a presynaptic Gi/Go-coupled auto- and heteroreceptor. Proxyfan was identified to be functionally selective, with different efficacies toward Gi/Go-dependent hH3R signaling pathways. However, the underlying molecular mechanism of functional selectivity of proxyfan is still unclear. In the current study, we investigated the role of different Gαi/o proteins in hH3R signaling, using a baculovirus/Sf9 cell expression system. We tested the hypothesis that ligand-specific coupling differences to defined Gi/Go-heterotrimers are responsible for functional selectivity of proxyfan at hH3R. In Sf9 membranes, full-length hH3R (445 amino acids) was expressed in combination with an excess of different mammalian G proteins (Gαi1, Gαi2, Gαi3, or Gαo1 and β1γ2 dimers, respectively). In addition, we constructed the fusion proteins hH3R-Gαi2 and hH3R-Gαo1 to ensure clearly defined receptor/G protein stoichiometries. Steady-state GTPase experiments were performed to directly measure the impact of each G protein on hH3R signal transduction. The hH3R coupled similarly to all G proteins. We also observed similar ligand-independent or constitutive activity. Proxyfan and various other imidazole-containing ligands, including full agonists, partial agonists, and inverse agonists, showed very similar pharmacological profiles not influenced by the type of G protein coexpressed. Selected ligands, examined in membranes expressing the fusion proteins hH3R-Gαi2 and hH3R-Gαo1 (plus β1γ2 dimers), yielded very similar results. Collectively, our data indicate that hH3R couples similarly to different Gαi/o-subunits and that ligand-specific active receptor conformations, resulting in G protein-coupling preferences, do not exist for proxyfan or other imidazole compounds investigated.
Footnotes
This work was supported by the Research Training Program (Graduiertenkolleg) [Grant GRK760] “Medicinal Chemistry: Molecular Recognition—Ligand-Receptor Interactions” of the German Research Foundation (to D.S.); the “Research Internships in Science and Engineering program of the German Academic Exchange service (Deutsche Akademische Austauschdienst) (to K.B. and J.T.); and COST Action [Grant BM0806] (to R.S. and D.S.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.162339.
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ABBREVIATIONS:
- H3R
- histamine H3 receptor
- GPCR
- G protein-coupled receptor
- h
- human
- β2AR
- β2-adrenoceptor
- H4R
- histamine H4 receptor
- aa
- amino acids
- PCR
- polymerase chain reaction
- JNJ-7753707
- (4-fluorophenyl)(1-methyl-2-an-1H-imidazol-5-yl)methanone
- GTPγS
- guanosine 5′-[γ-thio]triphosphate
- bp
- base pair(s)
- PAGE
- polyacrylamide gel electrophoresis
- r
- rat.
- Received October 4, 2009.
- Accepted December 2, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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