Molecular determinants for the high constitutive activity of the human histamine H₄ receptor: functional studies on orthologues and mutants

Abstract

BACKGROUND AND PURPOSESome histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4R) and rat H4 receptor (rH4R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M; double mutants: hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S. EXPERIMENTAL APPROACHSite-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([ KEY RESULTSConstitutive activity decreased from the hH4 receptor via the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. CONCLUSIONS AND IMPLICATIONSF169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs.

[3H]UR‐DE257: Development of a Tritium‐Labeled Squaramide‐Type Selective Histamine H2 Receptor Antagonist

“…2-3 drops Et 3 N were added and succinimidyl propionate (0.7-1.6 equiv), dissolved in CH 3 CN or MeOH, was added and the solution stirred at RT for 30 min to 18 h. The product was purified by preparative HPLC. gy with the previously reported protocols, [20,51,53,56] using Micro Beta2 1450 scintillation counter (PerkinElmer, Rodgau, Germany) in the 96-well plate format.…”

“…[52,53]) expressing the hH 2 R-G saS or the gpH 2 R-G saS and on the isolated spontaneously beating guinea pig right atrium [46,54] were performed as previously described, except that the incubation period of the guinea pig atrium in the presence of the antagonists was extended to 60 min. Binding data on recombinant histamine receptor subtypes and orthologues expressed in Sf9 cells (hH 1 R + RGS4; hH 2 R-G saS , gpH 2 R-G saS , rH 2 R-G saS ; hH 3 R + G ia2 + b 1 g 2 ; hH 4 R + G ia2 + b 1 g 2 ) or HEK293T CRE-Luc cells [55] (hH 2 R) using the following radioligands: H 1 R,[ …”

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

“…Amino acids F169 and S179 appear to play key roles for this high constitutive activity of the human H 4 receptor (Wifling et al, 2015). Because the human H 4 receptor exhibits high constitutive activity (Lim et al, 2005;Schneider et al, 2010b), H 4 receptor agonists decrease cAMP accumulation, whereas H 4 receptor inverse agonists increase cAMP accumulation (Lim et al, 2005;Kottke et al, 2011).…”

Aryl-1,3,5-triazine ligands of histamine H4 receptor attenuate inflammatory and nociceptive response to carrageen, zymosan and lipopolysaccharide

“…On the other hand, the species differences might be partially responsible for this effect. Some histamine H 4 R ligands act as inverse agonists at the human H 4 R, which is constitutively active, whereas as neutral antagonists at the constitutively inactive mouse and rat H 4 R [11]. Moreover, the test compounds and JNJ 7777120 may vary in their impact on β-arrestin-dependent intracellular pathway.…”

“…Thereby, the biological response in the absence of a bound ligand can be inhibited by H 4 R inverse agonists—the ligands that stabilize the inactive receptor conformation and are therefore able to reduce constitutive activity. It is important that primarily human H 4 R receptor possesses a high constitutive activity, whereas canine, murine and rat H 4 R orthologs show substantially lower activity in the absence of an agonist [11, 12]. …”