No Evidence for Functional Selectivity of Proxyfan at the Human Histamine H₃ Receptor Coupled to Defined G_i/G_o Protein Heterotrimers

Abstract

Numerous structurally diverse ligands were developed to target the human histamine H 3 receptor (hH 3 R), a presynaptic G i /G ocoupled auto-and heteroreceptor. Proxyfan was identified to be functionally selective, with different efficacies toward G i /G odependent hH 3 R 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 G␣ i/o proteins in hH 3 R signaling, using a baculovirus/Sf9 cell expression system. We tested the hypothesis that ligandspecific coupling differences to defined G i /G o -heterotrimers are responsible for functional selectivity of proxyfan at hH 3 R. In Sf9 membranes, full-length hH 3 R (445 amino acids) was expressed in combination with an excess of different mammalian G proteins (G␣ i1 , G␣ i2 , G␣ i3 , or G␣ o1 and ␤ 1 ␥ 2 dimers, respectively). In addition, we constructed the fusion proteins hH 3 R-G␣ i2 and hH 3 R-G␣ o1 to ensure clearly defined receptor/G protein stoichiometries. Steady-state GTPase experiments were performed to directly measure the impact of each G protein on hH 3 R signal transduction. The hH 3 R 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 hH 3 R-G␣ i2 and hH 3 R-G␣ o1 (plus ␤ 1 ␥ 2 dimers), yielded very similar results. Collectively, our data indicate that hH 3 R couples similarly to different G␣ i/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.

Comparison of the pharmacological properties of human and rat histamine H3-receptors

“…The antibody recognizing both species homologs was from GeneWay (San Diego, CA, USA). All other antibodies, purified G proteins, reagents for molecular biology, recombinant baculoviruses encoding mammalian G protein subunits, and the sources of test compounds were described before [23]. Chemical structures of H 3 R ligands are depicted in Fig.…”

“…Stock solutions (10 mM) of all H 3 R ligands described in this paper were prepared in distilled water and stored at -20°C. was synthesized as described [23]. Unlabeled nucleotides were from Roche (Indianapolis, IN, USA) and all other reagents were of the highest purity available and from standard suppliers.…”

“…The cDNA for the tagged rH 3 R protein was generated by sequential overlap-extension PCR in analogy to the procedure described recently for hH 3 R [23]. In the case of rH 3 R, the sense primer RAT HRH3-F-(5´-GAC GAT GAT GAC GCC ATG GAG CGC GCG CCG CC-3´) consisted of 15 bp of the 3'-end of SF and the first 17 bp of the 5'-end of the rH 3 R. The antisense primer RAT HRH3-RV (5´-GA TCC TCT AGA TTA GTG ATG GTG ATG ATG GTG CTT CCA GCA CTG CTC -3´) consisted of 15 bp of the C-terminus of the rH 3 R, and encoded a hexahistidine tag, the stop codon, and a Xba I site.…”

“…The protocols for virus amplification, protein expression and western blot analysis were described before [23]. Proteins transferred to nitrocellulose membranes were reacted with anti-hH 3 R (N-term) (1:1000), anti-rH 3 R (C-term) (1:1000) and anti-H 3 R (i3) (1:1000) Igs.…”

“…In this manner, only the number of functionally intact and heterologously expressed mammalian Gα i/o -subunits was quantitated. Experiments were performed in analogy to the assay described in Schnell et al [23]. Briefly, membranes were thawed, sedimented and resuspended in 10 mM Tris/HCl, pH 7.…”