JNJ7777120 selectively recruits β-arrestin to the histamine H4 receptor in a G protein-independent manner

Elizabeth M Rosethorne, Steven J Charlton. Novartis Institues for Biomedical Research, Horsham, United Kingdom.

The Gi/o-coupled histamine H4 receptor (H4R) is highly expressed in haemopoietic cells, suggesting H4R antagonists may provide therapeutic benefit in chronic inflammatory diseases. JNJ7777120 has been described previously as a novel, selective antagonist at the histamine H4R with Ki of 4 nM, and a pA2 equal to 8.1 in a cAMP-CRE reporter gene assay (Jablonowski et al. 2003; Thurmond et al. 2004). We have investigated the antagonistic properties of JNJ7777120 using two distinct downstream signalling readouts; G protein activation and β-arrestin recruitment.

PathHunter™ U2OS-H4/β-arrestin cells and membranes derived from this cell-line were evaluated in the DiscoveRx β-arrestin recruitment assay and a [35S]-GTPγS accumulation assay to investigate the antagonistic properties of JNJ7777120.

The H4R agonists histamine and clobenpropit increased β-arrestin recruitment to the H4R in a concentration-dependent manner. This β-arrestin recruitment could be inhibited by pre-incubation with thioperamide, a dual H3R / H4R antagonist, but not the H1R antagonist triprolidine. In contrast, pre-incubation with the H4R-selective antagonist JNJ7777120 did not antagonise, but rather potentiated the response to histamine. JNJ7777120 treatment alone resulted in an increase in β-arrestin recruitment, in a thioperamide-dependent manner. Schild analysis demonstrated competitive antagonism between thioperamide and both clobenpropit and JNJ7777120. Histamine and clobenpropit, but not JNJ7777120, were both able to induce [35S]-GTPγS accumulation in membranes prepared from U2OS-H4 cells. Thioperamide and JNJ7777120 were both able to inhibit [35S]-GTPγS accumulation induced by clobenpropit (see Table for summary data). All other histamine receptor agonists and antagonists tested had no effect in this assay format.

We have demonstrated that JNJ7777120 performs as an antagonist at the H4R for [35S]-GTPγS accumulation, and a partial agonist for β-arrestin recruitment. Histamine and clobenpropit have comparable potencies for both [35S]-GTPγS accumulation β-arrestin recruitment, suggesting little difference in the levels of receptor reserve between the two assays. In conclusion, JNJ7777120 selectively recruits β-arrestin to the H4R, seemingly independent of G protein activation.

Jablonowski JA, Grice CA, Chai WY et al., (2003) J. Med. Chem. 46, 3957-3960.
Thurmond RL, Desai PJ, Dunford PJ. et al., (2004) J Pharmacol Exp Ther 309, 404-413.