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

Background and purpose: Some histamine H4 receptor (H4R) ligands act as inverse agonists at the human H4R (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4R (mH4R) and rat H4R (rH4R). To study molecular determinants of constitutive activity, H4R 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 approach: Site-directed mutagenesis with pVL1392 plasmids containing hH4R or mH4R 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 ([3H]histamine) as well as in functional [35S]GTPγS assays with inverse, partial and full agonists of the hH4R. Key results: Constitutive activity decreased from the hH4R 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 hH4R. Partial inverse hH4R agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologs with lower or no constitutive activity. Some partial and full hH4R agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4R to M as in mH4R or A as in rH4R did not significantly reduce constitutive activity. Conclusions and implications: F169 and S179 are key amino acids for the high constitutive activity of the hH4R and may also be of relevance for other constitutively active GPCRs