Constitutive activity of the human histamine H4 receptor: Molecular modelling, binding and functional studies on wild-type and mutant H4R orthologs

The histamine H4R belongs to the class A of G-protein coupled receptors (GPCRs) and is considered as a promising drug target for the treatment of inflammatory diseases such as allergic asthma. The validation of the H4R in translational animal models is compromised by species-dependent differences regarding intrinsic activities, potencies and affinities of ligands, in particular, when comparing the hH4R (human) and the rodent orthologs, i. e., the mH4R (mouse) and rH4R (rat). In contrast to the mH4R and rH4R, the hH4R shows a high degree of constitutive activity. Therefore, H4R species orthologs represent ideal candidates to study the phenomenon of “constitutive activity”. As working hypothesis of the present project, the aforementioned species-dependent differences were supposed to be determined by one or several distinct amino acids in the ligand binding pocket of the human H4R compared to the orthologs of mouse and rat. Aiming at more detailed insights into the molecular determinants of ortholog-dependent ligand-receptor interactions, a series of H4R mutants were generated and expressed in Sf9 cells to determine and analyse radioligand binding and functional data ([35S]GTPγS assay) of selected ligands. In addition to F169, which was identified by Lim et al. as a key amino acid for distinct ligand binding affinities at H4R orthologs, suggested by molecular modelling studies, S179, S330 and R341 were replaced by the corresponding amino acids of the rodent H4Rs, resulting in hH4R-F169V, hH4R-S179M/A, hH4R-F169V+S179M/A, hH4R-S330R and hH4R-R341S. The reciprocal mH4R mutants, mH4R-V171F and mH4R-V171F+M181S, respectively, served as controls. Moreover, to study the role of the F168/F169 motif, which is also found in, e. g., the β2AR, H3R and the M2R, the mutated receptor protein hH4R-F168A was expressed in Sf9 cells. Additionally, R341 was replaced by the respective residue of the canine H4R, resulting in hH4R-R341E. Comparable expression levels and ratios of receptor to G-protein expression in Sf9 cell membranes were confirmed by [3H]histamine saturation binding and SDS-PAGE, followed by Coomassie staining and western blotting. Compared to the hH4R wild-type, especially UR-PI376, clozapine and isoloxapine revealed a significant decrease in potency and affinity at the hH4R-F169V single and the hH4R-F169V+A179M/A double mutants, respectively. The reverse mH4R mutants, mH4R-V171F and mH4R-V171F+M181S, respectively, became more hH4R-like. Moreover, the potency and/or affinity of most ligands was higher at the S179A than at the respective S179M mutants. Strikingly, the constitutive activity of the hH4R-F169V and the double mutants was significantly reduced compared to the wild-type hH4R. By contrast, an exchange of S179 by M or A alone did not significantly affect constitutive activity. The double mutants were comparable to the mH4R and to the rH4R, which are devoid of constitutive activity. The inverse agonism of thioperamide decreased from the hH4R via the hH4R-F169V mutant to the hH4R-F169V+S179M/A double mutants, respectively. The data for the hH4R-F168A mutant revealed a major contribution of F168 to ligand binding with a concomitant, up to over 100-fold decrease in ligand potencies and a complete loss of constitutive activity, compared to the wild-type hH4R. Thioperamide acted as a neutral antagonist and JNJ7777120 turned to partial agonism. Potencies and affinities of the ligands clozapine, isoloxapine and UR-PI376 slightly decreased at the hH4R-S330R mutant compared to the hH4R wild-type. Constitutive activities slightly decreased in case of the hH4R-S330R mutant. Compared to the hH4R, the affinity of UR-PI376 increased at the hH4R-R341E mutant. The constitutive activity of the hH4R-R341S mutant decreased slightly. Molecular modelling studies suggested that F168ECL2 and F169ECL2 interact with the surrounding hydrophobic and aromatic amino acids, which are supposed to be involved in the contraction of the binding pocket and, thus, in constitutive activity. S1795.43 was proposed to form an H-bond with T3236.55, which is precluded in case of mutation to M or A. S1795.43 alone was not the cause for the high constitutive activity of the hH4R. However, this amino acid in concert with F169ECL2 markedly contributes to the concomitant distal outward movement of TM5 and TM6. In conclusion, especially F168 and F169 alone or F169 in concert with S179 favour the conversion of the inactive to the active state of the human H4R. The fact that comparable amino acids are present in equivalent positions of other constitutively active GPCRs such as the β2AR or the H3R suggest a common mechanism of receptor activation