Homologous and heterologous phosphorylations of human histamine H1 receptor in intact cells.

Homologous and heterologous phosphorylations of histamine H1 receptor (H1R) in intact cells were investigated using Chinese hamster ovary cells stably co-expressing c-myc-tagged human histamine H1 and muscarinic M3 receptors. Increase in histamine-induced homologous phosphorylation of H1R was induced in a dose- and time-dependent manner. Maximum phosphorylation of H1R by 8-fold over the basal level was induced 1 min after the stimulation, and the increased phosphorylation level was maintained over 40 min. M3 receptor-mediated heterologous phosphorylation of H1R reached maximum by 2-fold over the basal level at 5 min after the stimulation and then rapidly returned to the basal level by 40 min after the stimulation. Histamine-induced phosphorylation of H1R was partially inhibited by three protein kinase inhibitors including Ro-31-8220 for protein kinase C (PKC), KN-93 for calcium/calmodulin-dependent kinase II (CaMKII), and KT5823 for protein kinase G (PKG), while, M3-receptor-mediated phosphorylation of H1R was completely inhibited by Ro 31-8220. Protein kinase activators including phorbol 12-myristate 13-acetate (PMA), 8-bromo-cyclic GMP (8-Br-cGMP), and 8-bromo-cyclic AMP (8-Br-cAMP) induced increases in H1R phosphorylation. Increased phosphorylation of H1R, by 5-fold over the basal level, induced with a combination of PMA, 8-Br-cGMP, and 8-Br-cAMP was still lower than that with histamine. It was suggested that H1R-mediated H1R phosphorylation involves the activation of PKC, CaMKII, PKG, and other unidentified kinases including G-protein coupled receptor kinases (GRKs) and that PKC is solely involved in M3 receptor-mediated H1R phosphorylation.