Histamine H2 Receptor Trafficking: Role of Arrestin, Dynamin, and Clathrin in Histamine H2 Receptor Internalization

Abstract

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with ␤-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.

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

“…As the crossdesensitization was independent not only of second messengers but also of downstream second messenger-dependent protein kinases, a possible cross-regulation at the receptor level was evaluated. 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).…”

“…In an attempt to determine whether a direct cause-effect existed between receptor cointernalization and crossdesensitization we used U937 derived clones (DC6, A2, and GB4) previously characterized in our laboratory that exhibit blockade of H2R internalization by different mechanisms. DC6 cell line was obtained by stable transfection with a dynamin dominant negative mutant which proved to dampen agonist-induced H2R endocytosis (Fernandez et al, 2008). A2 cells have a GRK2 antisense construct that diminishes GRK2 expression levels at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org and diminishes both H2R desensitization and internalization (Fernandez et al, 2002(Fernandez et al, , 2011.…”

“…The stably expressing human H1R and H2R cell lines (CHO-H1R and CHO-H2R) were previously generated in our laboratory (Notcovich et al, 2010;Tubio et al, 2010). DC6, A2, and GB4 cell lines were obtained by U937 stable transfection with a dynamin dominant negative mutant, a G protein-coupled receptor kinase 2 (GRK2) antisense construct, and a GRK2 dominant negative mutant of the kinase site, and were previously characterized (Fernandez et al, 2002(Fernandez et al, , 2008(Fernandez et al, , 2011.…”

International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors

“…Recently, the GTPase dynamin has been identified as a binding partner for the H 2 receptor, both in vitro and in vivo (Xu et al, 2008). A role of dynamin, b-arrestin, and clathrin in H 2 receptor 614 internalization has indeed also been reported (Fernandez et al, 2008).…”

“…Desensitization of the H 2 receptor is suggested to involve both GPCR kinase 2 and 3 (Rodriguez-Pena et al, 2000;Fernandez et al, 2011). The C-terminal tail is required for membrane targeting through a putative palmitoylation-mediated mechanism (Fukushima et al, 1997), and the last 51 amino acids are reported to be crucial for internalization (Fernandez et al, 2008). Recently, the GTPase dynamin has been identified as a binding partner for the H 2 receptor, both in vitro and in vivo (Xu et al, 2008).…”