Homologous and Heterologous Phosphorylations of Human Histamine H1 Receptor in Intact Cells

Abstract

Abstract. Homologous and heterologous phosphorylations of histamine H 1 receptor (H1R) in intact cells were investigated using Chinese hamster ovary cells stably co-expressing c-myctagged human histamine H 1 and muscarinic M 3 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. M 3 receptormediated 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, M 3 -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-BrcGMP), 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 M 3 receptor-mediated H1R phosphorylation.

Ubiquitin/proteasome‐dependent down‐regulation following clathrin‐mediated internalization of histamine H1‐receptors in Chinese hamster ovary cells

“…This is also in good accordance with recent findings that GRK2 is involved in the phosphorylation or desensitization of H 1 ‐receptors in CHO cells (Horio et al. , 2004; Miyoshi et al. , 2004), human embryonic kidney (HEK293) cells (Iwata et al.…”

“…These results are consistent with the evidence that protein kinase C is involved in the phosphorylation of H 1 ‐receptors to induce their down‐regulation but not internalization in CHO cells (Horio et al. , 2004; Miyoshi et al. , 2004), although there is controversial evidence against the involvement of protein kinase C in down‐regulation of H 1 ‐receptors (Smit et al.…”

“…CHO cells stably expressing human histamine H 1 ‐receptors (Miyoshi et al. , 2004) were cultured in 150‐cm 2 culture flasks as described previously (Hishinuma and Young, 1995).…”

Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-γ production in mice

“…Modulation of H 1 R signaling, like that of other G protein-coupled receptors, is complex and includes receptor desensitization, internalization, and subsequent downregulation (41,42). Desensitization of H 1 R is induced by both agonist-specific (homologous) and agonist nonspecific (heterologous) pathways, mainly involving PKC-mediated phosphorylation of H 1 R (43,44). PKC activation has previously been shown to inhibit H 1 R both at the protein level and at the gene expression level (45)(46)(47)(48).…”

Localized Expression of Histamine H1 Receptors in Syncytiotrophoblast Cells of Human Placenta

“…The [ 3 H]mepyramine binding assay was performed as described previously (20). A suspension of cell membranes (500 µg of protein) was incubated at 25°C for 60 min with various concentrations of [ Chinese hamster ovary (CHO) cells expressing c-myctagged human H 1 Rs (CHO-H 1 R cells) Expression of H 1 Rs in CHO cells was described previously (21,22). Briefly, the mammalian expression vector pdKCR-dhfr containing c-myc epitope-tagged human H 1 R gene was introduced into CHO cells that were deficient in dihydrofolate reductase [CHO(−) cells] using the calcium phosphate precipitation method.…”