C‐terminal of human histamine H₁receptors regulates their agonist‐induced clathrin‐mediated internalization and G‐protein signaling

Abstract

It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that G -protein-coupled human histamine H receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [ H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [ H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively.

Drug Hypersensitivity and Desensitizations: Mechanisms and New Approaches

“…Many studies have suggested that agonist-induced internalization of G-protein coupled receptors (GPCRs) from the cell surface into intracellular compartments regulates cellular responsiveness [58,59]. The multiple incremental suboptimal antigen doses administrated during DS may lead to the release of small amounts of histamine that would bind to receptors and subsequently induce internalization of the receptors.…”

Ca2+‐dependent down‐regulation of human histamine H1receptors in Chinese hamster ovary cells

“…In order to clarify whether H 1 receptor molecules are lost after the ionomycin treatment, we used CHO cells expressing human histamine H 1 receptors tagged with HA at the N terminus (Hishinuma et al . ), in which H 1 receptors were detected by an anti‐HA antibody instead of [ 3 H]mepyramine (Fig. ).…”

“…The binding of [ 3 H]mepyramine, a cell‐penetrant radioligand for H 1 receptors, to all cellular and cell‐surface H 1 receptors was detected, using mepyramine and pirdonium (a hydrophilic H 1 receptor ligand with a permanently charged quaternary ammonium group), respectively (Hishinuma et al . ). Briefly, aliquots (0.1 mL) of control and ionomycin‐treated cells (circa 3 × 10 5 cells, approximately 0.2–0.3 mg whole cell protein) were suspended in ice‐cold normal HEPES buffer (final volume 1 mL) and then incubated with 0.1–10 nM [ 3 H]mepyramine in the presence or absence of 10 μM mepyramine or 2 μM pirdonium at 4°C for 24 h in initial saturation experiments.…”

“…Histamine‐induced accumulation of [ 3 H]inositol phosphates was measured as described previously (Hishinuma et al . ). Briefly, cells were cultured on 10‐cm dishes and incubated with 0.12 μM myo‐[ 3 H]inositol (2.5 μCi/mL) for 24 h at 37°C.…”

“…CHO cells stably expressing human histamine H 1 receptors tagged with three molecules of HA: YPYDVPDYA at the N terminus (Hishinuma et al . ) were cultured on glass coverslips in six‐well plates and treated without (control) or with 10 μM ionomycin or 0.1 mM histamine at 37°C for 30 min in normal HEPES buffer. These cells were fixed with phosphate‐buffered saline containing 3% paraformaldehyde for 10 min and permeabilized with phosphate‐buffered saline containing 0.1% Triton‐X‐100 and 0.1% bovine serum albumin for 10 min.…”