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

Abstract

We investigated the regulatory pathways responsible for agonist-induced internalization and down-regulation of G q protein-coupled histamine H 1 -receptors in Chinese hamster ovary cells. Histamine-induced internalization and down-regulation of H 1 -receptors were detected as the loss of [ 3 H]mepyramine binding sites on intact cells accessible to hydrophilic and hydrophobic H 1 -receptor antagonists, pirdonium and mepyramine, respectively. Pretreatment of cells with 0.1 mM histamine for 30 min at 37°C induced internalization as well as down-regulation of H 1 -receptors, both of which were inhibited either in the presence of an inhibitor against G protein-coupled receptor kinases (ZnCl 2 ) or under hypertonic conditions where clathrin-dependent endocytosis is known to be inhibited, but were not affected by inhibitors against caveolae/raft-dependent endocytosis (filipin and nystatin). Down-regulation of H 1 -receptors, but not their internalization, was inhibited by protein kinase C inhibitors (chelerythrin or GF109203X), a ubiquitin E1 inhibitor (UBEI-41) and proteasome inhibitors (lactacystin and MG-132). Neither a Ca 2 + /calmodulin-dependent protein kinase II inhibitor (KN-62) nor lysosomal protease inhibitors (E-64, leupeptin, chloroquine and NH 4 Cl) affected the internalization and down-regulation of H 1 -receptors. These results suggest that H 1 -receptors internalize upon agonist stimulation via G protein-coupled receptor kinase/clathrin-dependent but caveolae/raft-independent mechanisms and are delivered to proteasomes, preferentially to lysosomes, for their prompt down-regulation.

The Affinity of Histamine for Gq Protein-Coupled Histamine H1-Receptors Is Predominantly Regulated by Their Internalization in Human Astrocytoma Cells

“…The results obtained in this study that histamine-induced internalization of H 1 -receptors was inhibited by ZnCl 2 suggest that GRK-mediated mechanisms may result in clathrin-dependent internalization of H 1 -receptors. These observations are consistent with our recent findings obtained in Chinese hamster ovary (CHO) cells on the involvement of GRK/clathrin-dependent mechanisms in histamineinduced internalization of H 1 -receptors (23). They are also in good accordance with the finding that GRK2 is (17,18), human embryonic kidney (HEK293) cells (20), and human myometrial smooth muscle cells (21), although human histamine H 1 -receptors, which were tagged with Myc at the Nterminal and green fluorescent protein (GFP) at the Cterminal, are reported to be internalized via clathrin-independent and raft-dependent mechanisms in CHO cells (19).…”

“…In sharp contrast to the predicted roles of GRKs in the internalization of H 1 -receptors, there is no evidence that other protein kinases, such as CaM kinase II and protein kinase C (PKC), are involved in histamine-induced internalization of H 1 -receptors (15,23); instead, phosphorylation of H 1 -receptors by CaM kinase II and PKC appears to mediate their subsequent down-regulation (17,18,23).…”

“…Since protein kinases, such as GRKs and/or Ca 2+ /CaM kinase II, are thought to be involved in the desensitization of histamine H 1 -receptors (13,16,20,21,23), we next examined the effects of ZnCl 2 and KN-62, which are known to inhibit GRKs (25 -27) and CaM kinase II (28), respectively, on histamine-induced changes in histamine binding to intact cells. Histamine-induced changes in displacement curves for histamine were completely inhibited by the presence of 0.4 mM ZnCl 2 , but not at all by the presence of 30 μM KN-62, during histamine pretreatment (Fig.…”

Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis

“…In fact, inhibition of proliferation was observed, indicating that the higher HA content in Y1 cells prevents the inhibition observed in H295R cells in the presence of exogenous HA. The endogenous HA content sustained over time could have triggered the internalization of its receptors, as reported in other systems, canceling responsiveness to HA (Miyoshi et al 2006, Hishinuma et al 2010. The lower number of HRH1 in Y1 cells than in H295R cells, calculated using the Scatchard analysis (1.2 vs 4.0 fmol/mg protein respectively), supports this hypothesis.…”

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

“…In a previous study performed in U937 cells endogenously expressing H2R as well as in COS7 and HEK293T cells transiently expressing H2R, we reported that the receptor is internalized following histamine or H2R-agonist stimulation as part of receptor trafficking (Fernandez et al, 2008). Furthermore, H1R is also internalized following histamine stimulation in H1R stably transfected CHO-K1 cells (Self et al, 2005;Hishinuma et al, 2010). Therefore internalization studies were performed in U937 and CHOtransfected cells to evaluate a putative reciprocal internalization of H1R and H2R.…”