Molecular and cellular analysis of aminergic G protein-coupled receptors: histamine H2, H4 and β2-adrenergic receptors, a scientific paradigm

About one third of the drugs currently available on the market address cell membrane-integrated G protein-coupled receptors (GPCRs). As such, the histamine H2 receptor (H2R) and the β2-adrenergic receptor (β2AR) have been successfully targeted in the treatment of frequently occurring gastric disorders and asthma, respectively. The novel histaminergic GPCR, the histamine H4 receptor (H4R), is currently in the focus of clinical trials as a promising target in the therapy of asthma and allergic rhinitis. However, being involved in many (patho)physiological functions, the H2R, H4R and β2AR gain great interest in academia and industry because of their potential to both, enable novel and to improve established therapeutic interventions. The first aim of this thesis was the dissection and comparison of the H2R- and β2AR-mediated effects on isolated human neutrophil granulocytes. Activation of both receptors on neutrophils results in an inhibition of inflammatory responses such as superoxide anion (O2•-) production, which could be therapeutically exploited. Although both, the H2R and the β2AR, preferentially couple to Gs proteins, a distinct behavior was observed in respect of their influence on cyclic adenosine 3',5'-monophosphate (cAMP) accumulation and O2•- production in neutrophils and a discrepancy with literature data, obtained in recombinant test systems, was noticed. In addition, ligand-directed signaling was detected, indicative of the concept of functional selectivity and suggesting that signaling events between the cAMP accumulation and the O2•- production are much more complex than assumed so far. Moreover, untypical behavior of H2R antagonists on neutrophils does not fit to the conventional understanding of antagonist/GPCR interaction. Possible reasons for described observations are discussed, although more detailed analyses of the signaling events in neutrophils are needed to mechanistically explain the divergence between the H2R and the β2AR and to better understand the multiplicity of GPCR-mediated signaling. The second aim of the present work was to characterize NG-acylated hetarylpropylguanidines, highly potent (partial) human H2R-agonists in Sf9 insect cell membranes, in physiologically more relevant human neutrophil granulocytes. In general, new H2R agonists are developed as valuable pharmacological tools and are regarded as novel drug candidates, e.g. in the therapy of acute myeloid leukemia. In contrast to histamine, the O2•- production by activated neutrophils was not affected by bivalent acylguanidines, whereas a monovalent ligand reduced O2•- production in a H2R-independent way. However, the ligands of interest stimulated cAMP accumulation in an H2R-dependent manner, albeit with drastically diminished agonistic activity compared with that in the respective recombinant test system. These results point to a drawback of acylguanidines, e.g. their amphiphilic character, and suggest that their drug-likeness needs to be further improved. Moreover, a contribution to the understanding of molecular interactions between the H2R and NG-acylated hetarylpropylguanidines was made. Mutagenesis studies revealed a rather marginal contribution of the extracelluar N-terminus to the species-selective profile of acylguanidines at the human H2R and the guinea pig H2R. Preclinical investigation of therapeutically interesting compounds in translational animal models is indispensable in drug research and development. Thus, identification and understanding of substantial pharmacological differences between H4R species orthologs at the molecular level is essential. In the third part of this thesis, the importance of extracellular regions for the species-specific pharmacology of the H4R was evaluated, using chimeric receptor approach and Sf9 insect cells as expression system. This study indicates that the N-terminus and the first extracellular loop are not responsible for the species differences between human and canine H4Rs. On the contrary, for the first time evidence is provided that the second and the third extracellular loop are involved in the H4R activation process. Mutagenesis studies revealed that low amino acid sequence homologies of the latter two loops contribute to the divergence between the inverse agonistic activity of JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) at the human and partial agonistic activity at the canine H4R, reported previously. Using a molecular dynamic simulation approach, suggestions about the involvement of the second and third extracellular loops in the inverse/partial agonistic behavior of JNJ7777120 are made at the molecular level