Analysis of Histamine H4R-Mediated Migration & Receptor Phosphorylation

SA Riaz1, SJ Charlton2, AB Tobin1. 1MRC Toxicology Unit/University of Leicester, Lancaster Road, Leicester LE1 9HN, UK, 2Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham RH12 5AB, UK

Histamine has an important role in the regulation of different physiological systems in the body. Chief among these is its role in the immune response. Mast cells, as well as being major producers of histamine, also express histamine receptors which drive various intracellular signalling pathways. Histamine receptors are prototypical G-protein coupled receptors (GPCRs) belonging to the Class A family. Histamine acting at the H4R of mast cells is known to mediate calcium mobilisation from intracellular stores in a pertussis toxin sensitive manner (Hofstra et al., 2003). We wanted to investigate the physiological response of mast cells in response to histamine and other H4R specific ligands.

Using HMC-1 cells, an immature mast cell line which lacks native expression of the human high-affinity IgE receptor (FcεRI) but contains other receptors such as c-kit, we have successfully shown the presence of the H4R using western blot analysis and immunocytochemistry, which corroborates previous studies (Lippert et al., 2004). We also show that histamine induces HMC-1 cell migration in a pertussis-sensitive and concentration-dependent manner confirming other studies (Hofstra et al., 2003). We show using specific ligands that migration at HMC-1 cells is not mediated by the other histamine receptors (H1-H2), but can be decreased in a concentration-dependent manner when antagonising the hH4R (Thioperamide and JNJ7777120, IC50= 4.68 μM and 2.07 μM, respectively). Interestingly, the IC50 value for thioperamide inhibition of HMC-1 cell chemotaxis in our assay seems to be comparable to other studies where HMC-1 cells have been used (1.0μM +0.5μM) (Hofstra et al., 2003), supporting the notion that the values obtained from our study are a true reflection of the action of thioperamide and JNJ7777120 on the H4R receptor in HMC-1 cells. JNJ7777120 has been described previously as a biased agonist at the H4R in PathhunterTM U2OS-H4/β-arrestin cells (Rosethorne & Charlton, 2011), acting as an inverse agonist to G-protein mediated signalling and a partial agonist for β-arrestin mediated signalling. Here we show that JNJ7777120 is acting as an inverse agonist in a physiologically relevant process as JNJ7777120 reduces basal levels of HMC-1 cell chemotaxis. This evidence would suggest that chemotaxis is a process mediated by G-protein signalling rather than β-arrestin-mediated signalling. Further work would involve looking at the phosphorylation of downstream effectors such as ERK to investigate the effects of JNJ7777120 in the HMC-1 cell line.

GPCRs or seven-transmembrane receptors have been shown to undergo phosphorylation in response to agonist occupation that mediates downstream signalling in the cell. To investigate these receptors and their phosphorylation sites, in vivo, our laboratory uses a proteomic approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This approach has allowed us to determine 10 sites either in the third intracellular loop or the C-terminal tail of hH4R which are phosphorylated. Six of these sites are seen to be agonist regulated. These results will be the basis for more sophisticated studies involving the phosphorylation sites of the hH4R, for example, investigating the concept of a phosphorylation “barcode” by raising phosphorylation-specific antibodies and using these to probe different cells and tissues known to endogenously express the hH4R and to investigate the effects of biased ligands acting at the receptor, for example, JNJ7777120.Hofstra CL et al, J Pharmacol Exp Ther 305(3): 1212, 2003. Lippert U et al, J Invest Dermatol 123(1):116 2004. Rosethorne EM & Charlton SJ, Mol Pharmacol 79(4):749, 2011. Rosethorne EM, & Charlton SJ (2011) Mol Pharmacol 79(4):749-57.