Shining light on the histamine H2 receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization.

So far, only little is known about the internalization process of the histamine H2 receptor (H2R). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type H2R agonists containing various fluorophores, heterocycles, and linkers (28-40) was synthesized. The ligands were pharmacologically characterized in several binding and functional assays. These studies revealed a significantly biased efficacy (Emax) for some of the compounds, e.g. 32: whereas 32 acted as strong partial (Emax: 0.77, mini-Gs recruitment) or full agonist (Emax: 1.04, [35S]GTPγS binding) with respect to G protein activation, it was only a weak partial agonist regarding ß-arrestin1/2 recruitment (Emax: 0.09-0.12) and failed to promote H2R internalization (confocal microscopy). On the other hand, H2R internalization was observed for compounds that exhibited moderate agonistic activity in the ß-arrestin1/2 pathways (Emax ≥ 0.22). The presented differently-biased fluorescent ligands are versatile molecular tools for future H2R studies on receptor trafficking and internalization e.g. using fluorescence microscopy.

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors.

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such "invasive" techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1-4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gßγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1-4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1-4Rs.