Isoform-specific biased agonism of histamine H3 receptor agonists

This article has not been copyedited and formatted. The final version may differ from this version. Abbreviations Imetit, 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate, iodoproxyfan, 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether, proxyfan, 4-[3-(benzyloxy)propyl]-1H-imidazole, NαMH, N-α-methylhistamine dihydrochloride, pERK, phosphorylated extracellular signal-regulated kinase, RαMH, (R)(−)-α-methylhistamine dihydrochloride This article has not been copyedited and formatted. The final version may differ from this version. Abstract The human histamine H 3 receptor (hH 3 R) is subject to extensive gene splicing that gives rise to a large number of functional and non-functional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signalling, this has not been studied for the H 3 R; further, it has unknown whether splice variants of the same receptor engender the same or differential biased signalling. Herein we have profiled the pharmacology of histamine receptor agonists at the two most abundant hH 3 R splice variants (hH 3 R 445 and hH 3 R 365) across seven signalling endpoints. Both isoforms engender biased signalling, notably for proxyfan (e.g. strong bias towards phosphorylation of GSK3β via the full-length receptor) and its congener iodoproxyfan, which are strongly consistent with the former's designation as a 'protean' agonist. The 80 amino acid IL3 deleted isoform, hH 3 R 365 is more permissive in its signalling than hH 3 R 445 : imetit, proxyfan and iodoproxyfan were all markedly biased away from calcium signalling and principal component analysis of the full dataset revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signalling between the two isoforms. Strikingly, hH 3 R 365 was completely unable to stimulate GSK3β phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signalling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants. This article has not been copyedited and formatted. The final version may differ from this version.