Agonist-Biased Signaling at the Histamine H₄ Receptor: JNJ7777120 Recruits β-Arrestin without Activating G Proteins

Abstract

The G i/o -coupled histamine H 4 receptor is highly expressed in hemopoietic cells and is a promising new target for the treatment of chronic inflammatory diseases. 1-[(5-Chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ7777120) has been described as a selective antagonist at the H 4 receptor and is widely used to characterize the physiological role of the H 4 receptor. We have investigated the pharmacological properties of JNJ7777120 using two distinct downstream signaling measurements, G protein activation and ␤-arrestin recruitment. The H 4 receptor agonists histamine and clobenpropit, but not JNJ7777120, were able to induce [35 S]GTP␥S binding in membranes prepared from U2OS-H 4 cells. Thioperamide, a dual H 3 /H 4 receptor antagonist, and JNJ7777120 were both able to inhibit the [ 35 S]GTP␥S binding induced by clobenpropit. Agonists and antagonists specific for other members of the histamine receptor family had no effect in this assay format. Histamine and clobenpropit increased ␤-arrestin recruitment to the H 4 receptor in a concentration-dependent manner. This ␤-arrestin recruitment could be inhibited by preincubation with thioperamide. We were surprised to find that preincubation with the H 4 -selective antagonist JNJ7777120 potentiated rather than antagonized the response to a submaximal concentration of clobenpropit. JNJ7777120 treatment alone resulted in an increase in ␤-arrestin recruitment, which again could be inhibited by preincubation with thioperamide. Schild analysis demonstrated competitive antagonism between thioperamide and both clobenpropit and JNJ7777120. Histamine and clobenpropit had comparable potencies for both [ 35 S]GTP␥S binding and ␤-arrestin recruitment, suggesting little difference in the levels of receptor reserve between the two assays. In conclusion, we have demonstrated that JNJ7777120 recruits ␤-arrestin to the H 4 receptor, independent of G protein activation.

Analysis of Multiple Histamine H4 Receptor Compound Classes Uncovers Gαi Protein- and β-Arrestin2-Biased Ligands

“…PathHunter U2OS ␤-arrestin2:EA cells stably expressing the human histamine H 4 receptor (U2OS-H 4 R) (Rosethorne and Charlton, 2011) were cultured in minimal essential medium containing L-glutamine supplemented with fetal bovine serum (10% v/v), penicillin (100 IU/ml), streptomycin (100 g/ml), G418 (Geneticin; 500 g/ml), and hygromycin (250 g/ ml) at 37°C and 5% CO 2 . One day before the ␤-arrestin2 recruitment assay, 10,000 cells/well were seeded in white, clear-bottomed 384-well ViewPlates (PerkinElmer Life and Analytical Sciences) in 20 l of minimal essential medium supplemented as described above and incubated at 37°C and 5% CO 2 .…”

“…Moreover, JNJ 7777120 induces ERK phosphorylation in a time-dependent manner typical for ␤-arrestin2-mediated signaling. In contrast, the inverse agonist thioperamide is not able to recruit ␤-arrestin2 but antagonizes the ␤-arrestin2 recruitment by both histamine and JNJ 7777120 (Rosethorne and Charlton, 2011). The ability of GPCR ligands to exhibit biased signaling is an emerging concept that has the potential to improve the efficacy and specificity and reduce the side effects of newly developed drugs (Galandrin et al, 2007;Kenakin, 2007;Rajagopal et al, 2010).…”

“…These reported hH 4 R activities are all pertussis toxin (PTx)-sensitive, indicating G␣ i -mediated signaling routes. In addition, as for many GPCR agonists, histamine stimulation of hH 4 R leads to recruitment of ␤-arrestin2 and downstream ERK phosphorylation (Rosethorne and Charlton, 2011). …”

International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors

“…Moreover, at canine H 4 receptor, thioperamide displayed weak partial agonism, whereas at human H 4 receptor, the compound was a strong inverse agonist, and at rat and mouse H 4 Histamine Receptors receptor, thioperamide is a neutral antagonist or very weak inverse agonist. To make matters more complicated, Rosethorne and Charlton (2011) expressed the human H 4 receptor in an osteosarcoma cell line and found that with respect to [ 35 S]GTPgS binding, JNJ-7777120 behaved as a neutral antagonist or very weak inverse agonist. In marked contrast, with respect to translocation of beta-arrestin to the plasma membrane, JNJ-7777120 exhibited strong partial agonistic activity.…”

A G Protein-Biased Ligand at theμ-Opioid Receptor Is Potently Analgesic with Reduced Gastrointestinal and Respiratory Dysfunction Compared with Morphine

“…Such a ligand, in a physiologic setting of an endogenous circulating agonist, can stimulate some receptor responses while inhibiting others (Violin et al, 2010;Kenakin, 2011). GPCR ligand bias has been observed between a number of intracellular signaling pathways, including coupling to different Ga subunits (Spengler et al, 1993;Eason et al, 1994), and between G protein coupling and b-arrestin recruitment (Azzi et al, 2003;Gesty-Palmer et al, 2006;DeWire et al, 2007;Violin and Lefkowitz, 2007;Kim et al, 2008;Ma et al, 2009;Gesty-Palmer and Luttrell, 2011;Rosethorne and Charlton, 2011). Evidence from knockout (KO) mice suggests that opioids signal through distinct MOR pathways, indicating that ligand bias might elicit differential MOR pharmacology.…”